OMV vaccines
09987345 ยท 2018-06-05
Assignee
Inventors
- Renata Maria Grifantini (Siena, IT)
- Oretta Finco (Siena, IT)
- Erika Bartolini (Siena, IT)
- Guido Grandi (Siena, IT)
Cpc classification
A61K2039/52
HUMAN NECESSITIES
A61K39/118
HUMAN NECESSITIES
C12N15/70
CHEMISTRY; METALLURGY
A61K2039/6037
HUMAN NECESSITIES
C12P21/02
CHEMISTRY; METALLURGY
International classification
A01N63/00
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A01N65/00
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
A61K45/00
HUMAN NECESSITIES
C12N15/70
CHEMISTRY; METALLURGY
A61K39/118
HUMAN NECESSITIES
C12P21/02
CHEMISTRY; METALLURGY
Abstract
The invention is in the field of outer membrane vesicles (OMV) and their uses. More particularly the present invention provides OMV obtained from a bacterium being an ompA mutant and/or a mutant in one or more components of the TolPal complex and presenting a heterologous antigen on its surface. The heterologous antigen is selected from the group consisting of Chlamydia trachomatis CT823, CT681, CT372, CT443, CT043, CT733, CT279, CT601 and CT153 for the treatment, prevention or diagnosis of Chlamydia infection.
Claims
1. A bacterium which is a OmpA (Outer membrane protein A) mutant and which presents a heterologous antigen on its surface; provided that where the bacterium is Escherichia coli (E.coli) BL21(DE3) it is a OmpAtolR mutant.
2. A bacterium according to claim 1, which does not express one or more components of the Tol-Pal complex.
3. A bacterium according to claim 2, which does not express TolR.
4. A bacterium according to claim 1, which expresses the Braun lipoprotein lpp.
5. A bacterium according to claim 1, wherein the bacterium is E. coli.
6. A bacterium according to claim 5, wherein the E. coli is E. coli BL21(DE3).
7. A bacterium according to claim 1, wherein the heterologous antigen is a membrane protein.
8. A bacterium according to claim 7, wherein the membrane protein is an outer membrane protein.
9. An outer membrane vesicle obtained from a bacterium according to claim 1, wherein the outer membrane vesicle expresses the heterologous antigen on its surface.
10. An outer membrane vesicle according to claim 9, which does not express the OmpA protein.
11. An outer membrane vesicle according to claim 9, which does not express the TolR protein.
12. An outer membrane vesicle according to claim 9, which expresses the Braun lipoprotein lpp.
13. An immunogenic composition comprising an outer membrane vesicle according to claim 9.
14. An immunogenic composition according to claim 13, which further comprises one or more additional antigens in a combined preparation for simultaneous, separate or sequential administration.
15. An immunogenic composition according to claim 14, wherein the one or more additional antigens are also presented by the outer membrane vesicle.
16. An immunogenic composition according to claim 14, wherein at least one of the one or more additional antigens is presented by a different outer membrane vesicle.
17. A method of raising an immune response in a mammal comprising administering an immunogenic composition according to claim 13 to the mammal.
18. The method of claim 17, wherein the immune response is a neutralising antibody response.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10) ) is TC0210pET BL21(DE3)TolR; (
) is TC0052pET BL21(DE3)TolR; (
) is TC0106pET BL21(DE3)TolR; (
) is TC0313pET BL21(DE3)TolR; (
) is TC0431pET B L21(DE3)TolR; (
) is TC0551pET BL21(DE3)TolR; (
) is TC0651pET BL21(DE3)TolR; (
) is TC0727pET BL21(DE3) TolR and) (
) is TC0890pET BL21(DE3)TolR.
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20) ), OMV-TC0210 (5 g) (
), OMV (50 g) (
), OMV-TC0210 (50 g) (
), and MOMP (
). Serum dilution is presented along the X axis and % of neutralisation is presented on the Y axis.
(21)
(22)
(23) ); with purified recombinant TC0210His (20 g) (
), with MOMP (20 g) (
) and with the OMVs alone (50 g) (
). Results are the mean of 6 independent experiments. Serum dilution is presented along the X axis and % of neutralisation is presented on the Y axis.
(24) ); OMV-TC0210 (50 g) (
); MOMP C. muridarum (20 g) (
); CT823-His (20 g) (
); TC0210-His (20 g) (
) and OMV (50 g) (
). Serum dilution is presented on the X axis and % of neutralisation is presented on the Y axis. The results are the mean of two experiments;
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
MODES FOR CARRYING OUT THE INVENTION
Example 1
Materials and Methods
(34) The following materials and methods are used in the examples unless stated otherwise:
(35) 1) Bacterial Strains, Cultures, and Reagents:
(36) Chlamydia muridarum strain Nigg and C. trachomatis serovar D strain D/UW-3/CX were grown on confluent monolayers of LLC-MK2 (ATCC CCL7) in Earle's minimal essential medium (EMEM) as described previously [195]; [196]. Purification of C. trachomatis and C. muridarum EBs was carried out by Renografin density gradient centrifugation as described previously [195]. Escherichia coli BL21(DE3) was grown aerobically in Luria broth (LB) medium (Difco) at 37 C. When appropriate, ampicillin (100 g/ml) and isopropyl--D-galactopyranoside (IPTG; 1 mM) were added to the medium. Unless specified, all chemicals used in this study were purchased from Sigma. Restriction enzymes and DNA modification enzymes were from New England Biolabs.
(37) 2) Gene Cloning and Protein Purification:
(38) To produce recombinant proteins such as CT823, TC0210, TC0727, TC0651, TC0313, TC0106, TC0551, TC0431, TC0890, CT681 (C. trachomatis MOMP [MOMP.sub.Ct]) and TC0052 (C. muridarum MOMP [MOMP.sub.Cm]), genes were PCR amplified from C. trachomatis and C. muridarum chromosomal DNA using specific primers annealing at the 5 and 3 ends of either gene and cloned into plasmid pET21b.sup.+ (Invitrogen) so as to fuse a six-histidine tag sequence at the 3 end. Cloning and purification of His fusions were performed as already described [196]. TC0727, TC0651, TC0106, TC0551, TC0431, TC0890, MOMP.sub.Ct and MOMP.sub.Cm expressed as His fusion proteins were purified from the insoluble protein fraction, while TC0313, CT823 and TC0210 expressed as His fusion proteins were purified from the soluble protein fraction according to the manufacturer's procedure.
(39) 3) Construction of BL21(DE3) toIR Deletion Mutant:
(40) The tolR mutant was produced by replacing tolR coding sequence with a kanamycin resistance (kmr) cassette. To this aim, a three-step PCR protocol was used to fuse the tolR upstream and downstream regions to the kmr gene. Briefly, the 528-bp upstream and 466-bp downstream regions of the tolR gene were amplified from E. coli BL21(DE3) genomic DNA with the specific primer pairs UpF (TCTGGAATCGAACTCTCTCG) (SEQ ID NO: 68)/UpR-kan (ATTTTGAGACACAACGTGGCTTTCATGGCTTACCCCTTGITG) (SEQ ID NO: 69); DownF-kan (TTCACGAGGCAGACCTCATAAACATCTGCCITTCCCITG) (SEQ ID NO: 70)/DownR (TTGCTTCTGCTTTAACTCGG) (SEQ ID 71), respectively. In parallel, the kmr cassette was amplified from plasmid pUC4K using the primers kan-F (ATGAGCCATATTCAACGGGAAAC) (SEQ ID NO: 72) and kan-R (TTAGAAAAACTCATCGAGCATCAAA) (SEQ ID NO: 73). Finally, the three amplified fragments were fused together by mixing 100 ng of each in a PCR containing the UpF/DownR primers. The linear fragment obtained, in which the kmr gene was flanked by the tolR upstream and downstream regions, was used to transform the BL21(DE3) E. coli strain (made electrocompetent by three washing steps in cold water), and tolR mutants were selected by plating transformed bacteria on Luria-Bertani (LB) plates containing 30 ug/ml of kanamycin.
(41) Recombination BL21(DE3) cells were produced by using the highly proficient homologous recombination system (red operon) [197]. Briefly, electrocompetent bacterial cells were transformed with 5 ug of plasmid pAJD434 by electroporation (5.9 ms at 2.5 kV). Bacteria were then grown for 1 h at 37 C. in 1 ml of SOC broth and then plated on LB plates containing trimethoprim (100 ug/ml). Expression of the red genes carried by pAJD434 was induced by adding 0.2% L-arabinose to the medium. The gene deletion of the tolR gene was confirmed by PCR. genomic DNA amplification using primers pairs UpF/Kan-R; Kan-F/Kan-R; Kan-F/DownR. The deletion was confirmed also using the primers tolR-F (CGGACCCGTATTCTTAAC) (SEQ ID NO: 74) and tolR-R (GCCTTCGCTTTAGCATCT) (SEQ ID NO: 75) annealing further upstream and downstream from the 5- and 3-flanking regions, respectively.
(42) 4) Construction of BL21(DE3) ompA Deletion Mutant:
(43) The ompA mutant was produced by replacing ompA coding sequence with a Chloramphenicol resistance (Cmr) cassette using specific primers. The procedure is the same as that utilized to produce BL21(DE3)tolR (see section 3) above). In particular, primers used to amplify the about 530 bp upstream and about 470 bp downstream regions of the ompA gene were amplified from BL21(DE3) genomic DNA with the specific primer pairs ompA_Up for: (GATCGGTTGGTTGGCAGAT) (SEQ ID NO: 76)/ompA cm_Up-rev: (CACCAGGATTTATTTATTCTGCGTTTTTGCGCCTCGTTATCAT) (SEQ ID NO: 77); ompA cm_Down for: (TACTGCGATGAGTGGCAGGCGCAGGCTTAAGTTCTCGTC) (SEQ ID NO: 78)/ompA Down rev: (AAAATCTTGAAAGCGGTTGG) (SEQ ID NO: 79); CMr FOR: (CGCAGAATAAATAAATCCTGGTG) (SEQ ID NO: 80)/CMr REV: (CCTGCCACTCATCGCAGTA) (SEQ ID NO: 81). Finally the three amplified fragments were fused together by mixing 100 ng of each in a PCR containing the ompA_Up for/ompA Down rev primers.
(44) The linear fragment obtained, in which the Cmr gene was flanked by the ompA upstream and downstream regions, was used to transform the BL21(DE3) E. coli strain (made electrocompetent by three washing steps in cold water). ompA mutants were selected by plating transformed bacteria on Luria-Bertani (LB) plates containing 20 ug/ml of Chloramphenicol.
(45) The gene deletion of the ompA gene was confirmed by PCR genomic DNA amplification using primers specifically annealing to Cmr cassette (CMr FOR/CMr REV), or ompA_Up for/CMr REV, or using primers specific for ompA in order to further verify the deletion of this gene (ompA FOR: (ATGAAAAAGACAGCTATCGC) (SEQ ID NO: 82)/ompA REV: (TTAAGCCTGCGGCTGAGTT) (SEQ ID NO: 83).
(46) 5) Expression of Chlamydial Antigens on BL21(DE3) tolR or on BL21(DE3) ompA:
(47) To express the 9 Chlamydia muridarum antigens (TC0052, TC0106, TC0210, TC0313, TC0431, TC0551, TC0651, TC0727, TC0890) on the outer membrane of E. coli mutant strains, genes coding for chlamydial antigens were fused in frame to the E. coli OmpA leader peptide. These fusions were then inserted in pET 21b (Invitrogen), a plasmid previously modified for cloning with the pipe method [198], by using specific primers annealing to the six-histidine tag sequence at the 3 end and to the gene coding for the E. coli OmpA leader peptide at the 5 end. The fusions were placed under the control of a lac promoter in the multicopy plasmid (pET). The obtained plasmid is called pET-TC0xyz. Plasmids were transformed in E. coli HK100 cells, made CaCl.sub.2 competent after several successive washes in cold, MgCl.sub.2 and CaCl.sub.2 solutions [198].
(48) Cells were plated on LB containing 100 ug/ml of Amp. at 37 C. O.N. Positives clones were grown in LB in order to produce mini preparations of plasmids (by using Qiagen mini prep kit).
(49) BL21(DE3) tolR and BL21 (DE3) ompA E. coli strain (made electrocompetent by three washing steps in cold water) were transformed with 10 ng of each pET-TC0xyz plasmid mini preparation. Bacteria were grown for 1 h at 37 C. in 1 ml of SOC broth and then plated on LB plates containing Amp. (100 ug/ml) and incubated O.N. at 37 C.
(50) 6) OMV Preparation:
(51) BL21(DE3)tolR and BL21(DE3)ompA E. coli cells expressing or not expressing chlamydial antigens were inoculated from fresh plate into 500 ml of LB (Luria Bertani broth)+Amp (100 ug/ml) and were incubated at 37 C. with shaking (200 r.p.m.) and growth. The induction of recombinant protein expression is made by addition of IPTG 0.1 mM at O.D.=0.4. Bacteria culture were grown until at 37 C. the O.D.=1. At that point, culture media were filtered through a 0.22 m pore-size filter (Millipore, Bedford, Mass.). The filtrates were subjected to high speed centrifugation (200,000g for 90 min), and the pellets containing the OMVs were washed with PBS and finally resuspended with PBS [199].
(52) 7) Western Blot Analysis:
(53) 20 ug of OMV preparations and 200 ng of TC0210 His were respectively size-separated by SDS-PAGE (4-12% gel) under reducing conditions and electroblotted onto nitrocellulose membranes. Membranes were saturated overnight with Milk Marwell 10% in PBS (phosphate Buffer) 0.1% Triton, shaking at 4 C. Then, membranes were incubated with specific mouse sera at RT for 2 hours (sera dilution 1:200). Anti mouse horseradish peroxidase conjugated IgG (Amersham Biosciences) was used as secondary antibody. Colorimetric staining was performed with the Opti 4CN substrate kit (Bio-Rad).
(54) 8) ELISA Assay:
(55) IgG directed to recombinant purified TC0210 were assayed by enzyme-linked immunosorbent assay (ELISA). Individual wells of micro-ELISA plates (Nunc Maxisorp) were coated with 1 g of recombinant protein in PBS (pH 7.4) at 4 C. overnight. The plates were washed, treated for 1 h at 37 C. with PBS-1% BSA, and 100 l aliquots of antisera at different serial dilutions in PBS-0.1% Tween were added to the wells. After incubation for 2 h at 37 C., plates were again washed and incubated for 1 h at 37 C. with alkaline-phosphatase conjugated goat anti-mouse IgG (Sigma) diluted 1:2500 in PBS-Tween. Thereafter 100 l of PNPP (Sigma) were added to the samples and incubated for 30 min. at room temperature. Optical densities were read at 405 nm and the sera-antibody titers were defined as the serum dilution yielding an OD value of 0.5.
(56) 9) Neutralization Assay:
(57) Sera obtained by immunizing mice (BALE/c or CD1) with OMV-TC0210 were tested in vitro for the neutralization activity. In vitro neutralization assays were performed on LLC-MK2 (Rhesus monkey kidney) epithelial cell cultures. Three serial dilutions of each sera pool were tested by diluting them 1:30, 1:90, 1:270 in Sucrose phosphate Buffer (SP). Also, purified infectious C. muridarum EB were diluted in the same Buffer to contain 310.sup.(5) IFU/ml, and 15 ul of EB suspension were added to each serum dilution in a final volume of 150 ul. Antibody-EB interaction was allowed by incubating for 30 min. at 37 C. Also, EB were incubated without sera as an infection control. 100 ul of each reaction mix, including EB diluted in SP without sera, were used to inoculate LLC-MK2 confluent monolayers (in duplicate for each serum dilution) in a 96-well tissue culture plate, and centrifuged at 2000 g for 1 hour at 37 C. After centrifugation, Earle's minimal essential medium containing Earle's salts, 10% fetal bovine serum and 1 ug/ml cycloheximide were added. Infected cultures were incubated at 37 C. for 24 hours, while in neutralization assays, in which EB of C. trachomatis were used for infection, infected cultures were incubated for 48 hours. Then, cell cultures were fixed by adding 100 ul of methanol for 5 minutes and the chlamydial inclusions were detected by staining with a mouse anti-Chlamydia fluorescein-conjugated monoclonal antibody (Merifluor Chlamydia, Meridian Diagnostics, Inc.). Finally, all inclusions for each well were counted at of 10 magnification.
(58) Calculations of the infectivity reduction by each sera pool were carried out using pre immune sera neutralization titers as basal levels.
(59) 10) Epitope Mapping:
(60) On a nitrocellulose membrane 95 synthetic peptides of TC0210 were spotted. Each peptide is constituted by 15 amino acids and overlaps 10mers with the following peptide. Three membranes, made with the same design, were washed with TBS (Tris-HCl 50 mM, NaCl 137 mM, KCl 2.7 mM) containing 0.05% of tween 20 (T-TBS), and then were incubated overnight at 4 C. with 2% milk Marwell in TBS (MBS). Then, on the three membranes, different sera pools (sera dilution 1:100) were tested respectively: sera of mice immunized with a) OMV expressing TC0210, b) TC0210His, and c) OMV alone.
(61) Finally, an anti-mouse horseradish peroxidase conjugated IgG (Amersham Biosciences) was used as a secondary antibody (sera dilution 1:5000). Colorimetric staining was performed with the Opti 4CN substrate kit (Bio-Rad).
Example 2
Construction of an E. coli BL21(DE3)tolR Deletion Mutant
(62) BL21 (DE3)tolR is a mutant E. coli strain in which the tolR mutation was introduced by replacing the tolR coding sequence with a Kanamycin resistance cassette. This strain is able to release a large quantity of outer membrane vesicles in the culture supernatant.
(63) It was found that the protein content released in the culture supernatant from the tolR mutant strain is higher compared to that released from the wild type strain (see
Example 3
Expression of Chlamydial Antigens in BL21(DE3)tolR or in BL21(DE3)ompA
(64) To allow presentation of each of the 9 Chlamydia muridarum antigens (TC0052, TC0106, TC0210, TC0313, TC0431, TC0551, TC0651, TC0727 and TC0890see Table 3 below) on the outer membrane of E. coli mutant strains, genes coding for chlamydial antigens were fused in frame to the E. coli OmpA leader peptide.
(65) TABLE-US-00003 TABLE 3 Chlamydia promising antigens C. trachomatis C. muridarum ANNOTATION homolog TC0052 MOMP CT681 TC0651 hypothetical protein CT372 TC0727 60 Kda Cystein Rich OMP CT443 TC0210 DO serine protease CT823 TC0313 hypothetical protein CT043 TC0106 hypothetical protein CT733 TC0551 Na(+)-translocating NADH-quinone CT279 reductase TC0431 MAC-Perforine protein CT153 TC0890 Invasine repeat family phosphatase CT601
(66) The fusions were placed under the control of a lac promotor in the multicopy plasmid pET21b+(Novagen). The obtained plasmid is called pET-TC0xyz.
(67) The 9 different pET-TC0xyz plasmids were transformed in E. coli BL21(DE3)tolR and E. coli BL21(DE3)ompA strains. The SDS PAGE gel analysis in
Example 4
OMV Preparation
(68) A schematic diagram of OMV preparation is shown in
Example 5
Analysis of OMV Preparations
(69) Western blot analysis was used to determine whether the OMV preparations expressed the heterologous antigens. Six out of the nine recombinant OMVs were shown to carry the heterologous proteins.
(70) However, in follow up experiments TC0431 was in fact shown to be expressed in the recombinant OMVs but at a low level as can be seen from
(71) The growth curve of E. coli BL21(DE3)tolR prepared in Example 3 expressing each of the 9 chlamydial antigens is shown in
(72) TC0210-OMV was found to be the best preparation in terms of yield and quantity of Chlamydia antigen that was expressed. Mass spectrometry confirmed the presence of the TC0210 peptide on the surface of TC0210-OMV preparations after the shaving of the same OMV preparation (the technique of Chlamydia shaving is described in WO 2007/110700 and a schematic diagram is shown in
(73) TABLE-US-00004 1 MMKRLLCVLLSTSVFSSPMLGYSAPKKDSSTGICLAASQS DRELSQEDLL 51 KEVSRGFSKVAAQATPGVVYIENFPKTGSQAIASPGNKRG FQENPFDYFN 101 DEFFNRFFGLPSHREQPRPQQRDAVRGTGFIVSEDGYVVT NHHVVEDAGK 151 IHVTLHDGQKYTAKIIGLDPKTDLAVIKIQAKNLPFLTFG NSDQLQIGDW 201 SIAIGNPFGLQATVTVGVISAKGRNQLHIVDFEDFIQTDA AINPGNSGGP 251 LLNIDGQVIGVNTAIVSGSGGYIGIGFAIPSLMAKRVIDQ LISDGQVTRG 301 FLGVTLQPIDSELAACYKLEKVYGALITDVVKGSPAEKAG LRQEDVIVAY 351 NGKEVESLSALRNAISLMMPGTRVVLKVVREGKFIEIPVT VTQIPAEDGV 401 SALQKMGVRVQNLTPEICKKLGLASDTRGIFVVSVEAGSP AASAGVVPGQ 451 LILAVNRQRVSSVEELNQVLKNAKGENVLLMVSQGEVIRF VVLKSDE
(74) TC0210 has the properties that are shown in Table 4.
(75) TABLE-US-00005 TABLE 4 TC0210 C. muridarum/ C. muridarum/ C. trachomatis C. trachomatis Protein name/ antigen % Similarity annotation Novartis scientific data Other data from literature TC0210/CT823 91.8 HtrA/DO serine Surface exposed Serine endoprotease, protease Antibody and CD4-th1 temperature-activated shows both inducer in mouse and chaperon and protease activities humans (Huston et al., FEBS Lett., 2007); Highly homologous among Immunogenic in humans the 8 major serovars (Sanchez-Campillo et al., Electrophoresis, 1999)
(76) A sequence alignment of C. muridarum TC0210 with C. trachomatis CT823 is provided in
(77)
(78) The analysis of the protein chip shown in
(79) This is supported by
(80)
(81) Recombinant HtrA and the native HtrA contained in the C. muridarum EBs was also recognized by a mouse polyclonal antibody serum which has been prepared by immunizing mice with OMV preparation from BL21(DE3)TolR TC0210 as shown by Western Blot in
(82) The FACS analysis described above for
Example 6
Immunization of BALB Mice with OMV-TC0210
(83) Mice were immunised according to the schedule shown in
(84) Western Blot and ELISA Assay
(85) Following immunisation of the mice, the mice sera were tested by Western blot analysis and ELISA in order to evaluate the production of anti-TC0210 antibodies. For each Western blot, the purified C. muridarum recombinant protein TC0210 was loaded.
(86) The ELISA results of
(87) Neutralization of Infection with E.B. Of C. muridarum.
(88) There is much evidence to support an important role for neutralizing antibodies in the protection against Chlamydia infection. In order to evaluate this, a neutralization assay was performed using sera of immunized mice with the OMV preparation expressing the chlamydial antigen TC0210. The results are shown in
(89) Sera of mice immunized with 50 g OMVs expressing TC0210 (Group 5 mice) are able to neutralize in vitro C. muridarum infection with a titer of 1:90 (the neutralization titer is defined as the serum dilution able to reduce EB infection by 50%) (see () line). This sera is almost as potent at neutralising C. muridarum in vitro as the sera obtained by immunizing mice with purified recombinant MOMP (positive controlsee the (
) line). In contrast, sera of mice immunized with purified recombinant TC0210 are not able to neutralize the C. muridarum infection; in fact neutralization percentages are very low also at minimal serum dilution (1:30) (see the (
) line). The (
) line shows the percentage of neutralization relative to OMV without chlamydial antigens. Thus, the neutralisation percentage for purified recombinant TC0210 is very similar for the neutralisation percentage obtained for OMV alone. These calculations have been done versus pre-immune sera. This is one of the first examples in which antibodies directed against a chlamydial antigen, other than MOMP, have been able to neutralize chlamydial infectivity in vitro. Surprisingly, these data show that the TC0210 antigen, which is not protective when tested in a chlamydial animal model when administered in its purified form, becomes protective when presented in an OMV of the invention.
(90) The neutralisation assay was repeated again and the results are shown in
(91) Neutralization of Infection with E.B. Of C. trachomatis.
(92) CT823 is the C. trachomatis homologous protein to TC0210. The ability of the anti OMV-TC0210 sera to neutralize in vitro infection by Chlamydia trachomatis was tested.
(93) Purified C. trachomatis EBs were incubated with mouse sera at three different dilutions at 37 C. for 30 min. Residual infectivity was determined on LLC-MK2 cells by counting IFU/cs. Neutralization percentages were measured in two independently performed neutralization assays and calculated versus preimmune sera ) line) is able to neutralize C. trachomatis efficiently in vitro (with a similar titer observed by infecting with E.B. of C. muridarum). Indeed, anti-OMV-TC0210 mouse polyclonal serum was found to be almost as potent at neutralising C. trachomatis infectivity as the sera of mice immunised with MOMP from C. trachomatis (see the (
) line, which is the positive control). The neutralizing percentage of this serum is also higher than that obtained after immunization with the homologue recombinant purified protein of C. trachomatis, CT823 (see (
) line). The (
) line represents the neutralizing percentage of sera immunized with MOMP of C. muridarum; this serum does not neutralize the chlamydial infection. Likewise, sera of mice immunized with OMV (see (
) line) or with TC0210His (see (
) line) did not neutralize the chlamydial infection. It was found that OMV-TC0210 specific-antibody neutralises C. trachomatis infection in vitro at the same level observed for C. muridarum. Thus, an immune response raised against the C. muridarum antigen may also neutralise infection against C. trachomatis.
Example 7
Immunization of CD1 Mice with OMV-TC0210
(94) In order to confirm the neutralization results of Example 6, immunization was repeated in CD1 mice (5-6 weeks old). Groups of CD1 mice (5 mice in each group) were immunised according to the following immunisation scheme: Group 1: 50 g OMV+Alum; Group 2: 50 g OMV-TC0210+Alum; Group 3: 20 g TC0210His+50 g OMV+Alum; Group 4: 1 g TC0210His+50 g OMV+Alum; Group 5: 1 g TC0210His+Alum; Group 6: 20 g TC0210His+Alum. The scheme was devised also to test whether there is an adjuvant effect of OMV (see Groups 3 and 4). Mice were immunized on days 1, 20 and 40. Sera were collected for the neutralization assay on day 60 (see
(95) Immunogenicity Comparison Between TC0210 Expressed on the OMV and the Recombinant Form.
(96) Western blots were performed using sera of mice immunized with 50 g of OMV-TC0210 (Group 2) and sera of mice immunized with 1 g of TC0210His plus 50 g of OMV without chlamydial antigens (Group 4), in order to compare the immunogenicity between TC0210 expressed on the OMV and the recombinant form. The quantity of chlamydial antigen present on the E. coli OMV surface is estimated to be 1% of the total content of E. coli proteins (about 0.5 ug of chlamydial protein in 50 ug of OMVs).
(97) The Western blot results of
(98) The Western Blot results of
(99) ELISA Titers
(100)
(101) From left to right, the bars in
(102) Although antibody titres of sera from mice immunised with OMVs expressing TC0210 are lower than those obtained with TC0210His or with the same recombinant protein+OMVs, the antibodies that are present in sera from mice immunised with OMVs expressing TC0210 are better in terms of neutralizing activity (see below).
(103) Although not yet investigated, it is reasonable that the ELISA results would have been different if Chlamydia EBs had been used for the coating, as these include TC0210 in its native conformation, instead of the recombinant protein as used in the present experiment. In fact, a coating with EBs would allow the detection of antibodies raised against conformational epitopes.
(104) Neutralisation Assay
(105) ), with 20 g of TC0210-His purified protein (Group 6:
). Sera of mice immunized with OMV alone (Group 1:
) and with recombinant TC0210-His (1 g) plus OMV alone (Group 4:
), were included as negative controls, while sera of mice immunized with recombinant MOMP (
) were used as the positive control. Data shown are the means and standard deviations of 12 samples.
(106) Sera of mice immunized with OMV expressing TC0210 () were found to neutralize the Chlamydial muridarum infection as efficiently as MOMP (
), while sera of mice immunized with OMV without chlamydial antigen or TC0210His do not.
(107) Also the neutralization results indicate that OMVs do not have an adjuvant effect. Instead, the result is due to the capability of recombinant OMV-TC0210 to present the heterologous antigen in its natural conformation and composition.
Example 8
Epitope Mapping Analysis on TC0210
(108) Epitope mapping experiments were performed in order to verify if there were some differences in terms of linear epitopes recognized between sera of mice immunized with OMV-TC0210 and sera of mice immunized with TC0210His. 95 overlapping synthetic peptides covering the full length of the TC0210 antigen were spotted on three membranes, respectively. Each peptide is constituted by 15 amino acids and overlaps 10mers with the following peptide. On the three membranes, different sera pools were tested: sera of mice immunized a) with OMV expressing TC0210, b) with TC0210His, c) with OMV alone (sera of mice immunized with OMV without the chlamydial antigen were used as a negative control), respectively. An anti-mouse horseradish peroxidase conjugated IgG was used as the secondary antibody.
(109)
(110) Sera of mice immunised with TC0210His recognised the following epitope with high specificity: NKRGFQENPFDYFNDEFFNRFFGLP (SEQ ID NO: 84). Sera of mice immunised with TC0210His recognised the following epitope with medium specificity: SHREQ.
(111) The epitope GENVLLMVSQGEVIR (SEQ ID NO: 55) indicated inside the box on the OMV-TC0210 membrane of
(112) It is hypothesized that sera produced by immunising with OMV-TC0210 and sera produced by immunising with TC0210 would be able to recognise the matching epitopes in CT823. Thus, it is hypothesised that the sera of mice immunized with OMV-TC0210 would presumably recognise the following CT823 epitope with high specificity: DYFNDEFFNRFFGLP (SEQ ID NO: 56). The sera of mice immunised with OMV-TC0210 would presumably recognise the following CT823 epitopes with medium specificity: SHREQ (SEQ ID NO: 57), ALQKMGVRVQNITPE (SEQ ID NO: 58) and NQVLKNSKGENVLLM (SEQ ID NO: 59). The sera of mice immunised with OMV-TC0210 would presumably recognise the following CT823 epitopes with low specificity: SPMLGYSASKKDSKADICLA (SEQ ID NO: 60), EDLLKEVSRGFSRVAAKATP (SEQ ID NO: 61), TGNQAIASPGNKRGFQENPF (SEQ ID NO: 62), IAIGNPFGLQATVTVGVISAKGRNQLHIVD (SEQ ID NO: 63) and NTAIVSGSGGYIGIGFAIPSLMAKRVIDQL (SEQ ID NO: 64). Sera of mice immunised with TC0210-His would presumably recognise the following CT823 epitope with high specificity: DYFNDEFFNRFFGLP. Sera of mice immunised with TC0210-His would presumably recognise the following CT823 epitope with medium specificity: SHREQ (SEQ ID NO: 57).
Example 9
Increasing the Quantity of Outer Membrane Proteins on the OMV
(113) OmpA is involved in the structural maintenance of the membrane system. Probably for this reason, the absence of this protein destabilizes the bacterial outer membrane resulting in the release of an abundant quantity of OMV. Release of OMV in the culture supernatant of BL21(DE3)ompA mutant strain was observed here. The OMV preparation has been made as previously described and shown in the schematic diagram of
(114) It was surprisingly found that all proteins present in this new OMV preparation are outer membrane proteins. This result underlines that the quality of the new OMV preparation is better than that obtained from the E. coli BL21(DE3)tolR mutant strain, in which some cytoplasmic proteins were also found. Specifically, in the E. coli BL21(DE3)tolR mutant strain, only about 75% of the 100 OMV proteins were outer membrane proteins.
Example 10
Expression of Chlamydial Antigens in the BL21(DE3)ompA Mutant Strain
(115) Chlamydial antigens were expressed in the E. coli BL21(DE3)ompA strain in order to verify if there is an increase in the quantity of chlamydial antigens in the derived recombinant OMV.
(116) OMVs were prepared from culture supernatants of BL21(DE3)ompA strain expressing TC0210 as previously described and shown in the schematic diagram of
(117) As shown in
(118) TABLE-US-00006 (SEQIDNO:46) VAAQATPGVVYIENFPK, (SEQIDNO:47) GFQENPFDYFNDEFFNRFFGLPSHREQPRPQQR, (SEQIDNO:48) GTGFIVSEDGYVVTNHHVVEDAGK, (SEQIDNO:49) TDLAVIKIQAK, (SEQIDNO:50) VIDQLISDGQVTR, (SEQIDNO:51) AGLRQEDVIVAYNGKEVESLSALR, (SEQIDNO:52) FIEIPVTVTQIPAEDGVSALQK, (SEQIDNO:53) VQNLTPEICK, and (SEQIDNO:54) NAKGENVLLMVSQGEVIR.
(119) The inventors have surprisingly found that BL21(DE3)ompA mutant strains generate an increased quantity of heterologous antigen on their OMVs relative to OmpA wild type strains. OmpA is the most abundant protein on E. coli outer membrane. The inventors have found that, in the OMV, the deletion of this protein improves the relative abundance of chlamydial antigen with respect to the E. coli outer membrane proteins.
(120) This increased amount of expressed chlamydial antigen suggests that OMVs from ompA strains, for example, BL21(DE3)ompA-TC0210, are good candidates for raising neutralizing antibodies in mice.
(121) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.
(122) TABLE-US-00007 SEQUENCE SEQIDNO:1-CT823proteinsequence MMKRLLCVLLSTSVFSSPMLGYSASKKDSKADICLAVSSGDQEVSQEDLLKEVSRGFSRVAAKATPGVVYI ENFPKTGNQAIASPGNKRGFQENPFDYFNDEFFNRFFGLPSHREQQRPQQRDAVRGTGFIVSEDGYVVTNH HVVEDAGKIHVTLHDGQKYTAKIVGLDPKTDLAVIKIQAEKLPFLTFGNSDQLQIGDWAIAIGNPFGLQAT VTVGVISAKGRNQLHIVDFEDFIQTDAAINPGNSGGPLLNINGQVIGVNTAIVSGSGGYIGIGFAIPSLMA KRVIDQLISDGQVTRGFLGVTLQPIDSELATCYKLEKVYGALVTDVVKGSPAEKAGLRQEDVIVAYNGKEV ESLSALRNAISLMMPGTRVVLKIVREGKTIEIPVTVTQIPTEDGVSALQKMGVRVQNITPEICKKLGLAAD TRGILVVAVEAGSPAASAGVAPGQLILAVNRQRVASVEELNQVLKNSKGENVLLMVSQGDVVRFIVLKSDE SEQIDNO:2-CT823nucleotidesequence ATGATGAAAAGATTATTATGTGTGTTGCTATCGACATCAGTTTTCTCTTCGCCAATGCTAGGCTATAGTGC GTCAAAGAAAGATTCTAAGGCTGATATTTGTCTTGCAGTATCCTCAGGAGATCAAGAGGTTTCACAAGAAG ATCTGCTCAAAGAAGTATCCCGAGGATTTTCTCGGGTCGCTGCTAAGGCAACGCCTGGAGTTGTATATATA GAAAATTTTCCTAAAACAGGGAACCAGGCTATTGCTTCTCCAGGAAACAAAAGAGGCTTTCAAGAGAACCC TTTTGATTATTTTAATGACGAATTTTTTAATCGATTTTTTGGATTGCCTTCGCATAGAGAGCAGCAGCGTC CGCAGCAGCGTGATGCTGTAAGAGGAACTGGGTTCATTGTTTCTGAAGATGGTTATGTTGTTACTAACCAT CATGTAGTCGAGGATGCAGGAAAAATTCATGTTACTCTCCACGACGGACAAAAATACACAGCTAAGATCGT GGGGTTAGATCCAAAAACAGATCTTGCTGTGATCAAAATTCAAGCGGAGAAATTACCATTTTTGACTTTTG GGAATTCTGATCAGCTGCAGATAGGTGACTGGGCTATTGCTATTGGAAATCCTTTTGGATTGCAAGCAACG GTCACTGTCGGGGTCATTAGTGCTAAAGGAAGAAATCAGCTACATATTGTAGATTTCGAAGACTTTATTCA AACAGATGCTGCCATTAATCCTGGGAATTCAGGCGGTCCATTGTTAAACATCAATGGTCAAGTTATCGGGG TTAATACTGCCATTGTCAGTGGTAGCGGGGGATATATTGGAATAGGGTTTGCTATTCCTAGCTTGATGGCT AAACGAGTCATTGATCAATTGATTAGTGATGGGCAGGTAACAAGAGGCTTTTTGGGAGTTACCTTGCAACC GATAGATTCTGAATTGGCTACTTGTTACAAATTGGAAAAAGTGTACGGAGCTTTGGTGACGGATGTTGTTA AAGGTTCTCCAGCAGAAAAAGCAGGGCTGCGCCAAGAAGATGTCATTGTGGCTTACAATGGAAAAGAAGTA GAGTCTTTGAGTGCGTTGCGTAATGCCATTTCCCTAATGATGCCAGGGACTCGTGTTGTTTTAAAAATCGT TCGTGAAGGGAAAACAATCGAGATACCTGTGACGGTTACACAGATCCCAACAGAGGATGGCGTTTCAGCGT TGCAGAAGATGGGAGTCCGTGTTCAGAACATTACTCCAGAAATTTGTAAGAAACTCGGATTGGCAGCAGAT ACCCGAGGGATTCTGGTAGTTGCTGTGGAGGCAGGCTCGCCTGCAGCTTCTGCAGGCGTCGCTCCTGGACA GCTTATCTTAGCGGTGAATAGGCAGCGAGTCGCTTCCGTTGAAGAGTTAAATCAGGTTTTGAAAAACTCGA AAGGAGAGAATGTTCTCCTTATGGTTTCTCAAGGAGATGTGGTGCGATTCATCGTCTTGAAATCAGACGAG TAG SEQIDNO:3-CT733nucleotidesequence ATGTTAATAAACTTTACCTTTCGCAACTGTCTTTTGTTCCTTGTCACACTGTCTAGTGTCCCTGTTTTCTC AGCACCTCAACCTCGCGGAACGCTTCCTAGCTCGACCACAAAAATTGGATCAGAAGTTTGGATTGAACAAA AAGTCCGCCAATATCCAGAGCTTTTATGGTTAGTAGAGCCGTCCTCTACGGGAGCCTCTTTAAAATCTCCT TCAGGAGCCATCTTTTCTCCAACATTATTCCAAAAAAAGGTCCCTGCTTTCGATATCGCAGTGCGCAGTTT GATTCACTTACATTTATTAATCCAGGGTTCCCGCCAAGCCTATGCTCAACTGATCCAACTACAGACCAGCG AATCCCCTCTAACATTTAAGCAATTCCTTGCATTGCATAAGCAATTAACTCTATTTTTAAATTCCCCTAAG GAATTTTATGACTCTGTTAAAGTGTTAGAGACAGCTATCGTCTTACGTCACTTAGGCTGTTCAACTAAGGC TGTTGCTGCGTTTAAACCTTATTTCTCAGAAATGCAAAGAGAGGCTTTTTACACTAAGGCTCTGCATGTAC TACACACCTTCCCAGAGCTAAGCCCATCATTTGCTCGCCTCTCTCCGGAGCAGAAAACTCTCTTCTTCTCC TTGAGAAAATTGGCGAATTACGATGAGTTACTCTCGCTGACGAACACCCCAAGTTTTCAGCTTCTGTCTGC TGGGCGCTCGCAACGAGCTCTTTTAGCTCTGGACTTGTACCTCTATGCTTTGGATTCCTGTGGAGAACAGG GGATGTCCTCTCAATTCCACACAAACTTCGCACCTCTACAGTCCATGTTGCAACAATACGCTACTGTAGAA GAGGCCTTTTCTCGTTATTTTACTTACCGAGCTAATCGATTAGGATTTGATGGCTCTTCTCGATCCGAGAT GGCTTTAGTAAGAATGGCCACCTTGATGAACTTGTCTCCTTCCGAAGCTGCGATTTTAACCACAAGCTTCA AAACCCTTCCTACAGAAGAAGCGGATACTTTGATCAATAGTTTCTATACCAATAAGGGCGATTCGTTGGCT CTTTCTCTGCGAGGGTTGCCTACACTTGTATCCGAACTGACGCGAACTGCCCATGGCAATACCAATGCAGA AGCTCGATCTCAGCAAATTTATGCAACTACCCTATCGCTAGTAGTAAAGAGTCTGAAAGCGCACAAAGAAA TGCTAAACAAGCAAATTCTTTCTAAGGAAATTGTTTTAGATTTCTCAGAAACTGCAGCTTCTTGCCAAGGA TTGGATATCTTTTCCGAGAATGTCGCTGTTCAAATTCACTTAAATGGAACCGTTAGTATCCATTTATAA SEQIDNO:4-CT733proteinsequence MLINFTFRNCLLFLVTLSSVPVFSAPQPRGTLPSSTTKIGSEVWIEQKVRQYPELLWLVEPSSTGASLKSP SGAIFSPTLFQKKVPAFDIAVRSLIHLHLLIQGSRQAYAQLIQLQTSESPLTFKQFLALHKQLTLFLNSPK EFYDSVKVLETAIVLRHLGCSTKAVAAFKPYFSEMQREAFYTKALHVLHTFPELSPSFARLSPEQKTLFFS LRKLANYDELLSLTNTPSFQLLSAGRSQRALLALDLYLYALDSCGEQGMSSQFHTNFAPLQSMLQQYATVE EAFSRYFTYRANRLGFDGSSRSEMALVRMATLMNLSPSEAAILTTSFKTLPTEEADTLINSFYTNKGDSLA LSLRGLPTLVSELTRTAHGNTNAEARSQQIYATTLSLVVKSLKAHKEMLNKQILSKEIVLDFSETAASCQG LDIFSENVAVQIHLNGTVSIHL SEQIDNO:5-CT153nucleotidesequence ATGACTAAGCCTTCTTTCTTATACGTTATTCAACCTTTTTCCGTATTTAATCCACGATTAGGACGTTTCTC TACAGACTCAGATACTTATATCGAAGAAGAAAACCGCCTAGCATCGTTCATTGAGAGTTTGCCACTGGAGA TCTTCGATATACCTTCTTTCATGGAAACCGCGATTTCCAATAGCCCCTATATTTTATCTTGGGAGACAACT AAAGACGGCGCTCTGTTCACTATTCTTGAACCCAAACTCTCAGCTTGCGCAGCCACTTGCCTGGTAGCCCC TTCTATACAAATGAAATCCGATGCGGAGCTCCTAGAAGAAATTAAGCAAGCGTTATTACGCAGCTCTCATG ACGGTGTGAAATATCGCATCACCAGAGAATCCTTCTCTCCAGAAAAGAAAACTCCTAAGGTTGCTCTAGTC GATGACGATATTGAATTGATTCGCAATGTCGACTTTTTGGGTAGAGCTGTTGACATTGTCAAATTAGACCC TATTAATATTCTGAATACCGTAAGCGAAGAGAATATTCTAGATTACTCTTTTACAAGAGAAACGGCTCAGC TGAGCGCGGATGGTCGTTTTGGTATTCCTCCAGGGACTAAGCTATTCCCTAAACCTTCTTTTGATGTAGAA ATCAGTACCTCCATTTTCGAAGAAACAACTTCATTTACTCGAAGTTTTTCTGCATCGGTTACTTTTAGTGT ACCAGACCTCGCGGCGACTATGCCTCTTCAAAGCCCTCCCATGGTAGAAAATGGTCAAAAAGAAATTTGTG TCATTCAAAAACACTTATTCCCAAGCTACTCTCCTAAACTAGTCGATATTGTTAAACGATACAAAAGAGAG GCTAAGATCTTGATTAACAAGCTTGCCTTTGGAATGTTATGGCGACATCGGGCTAAAAGCCAAATCCTCAC CGAGGGAAGCGTACGTCTAGACTTACAAGGATTCACAGAATCGAAGTACAATTACCAGATTCAAGTAGGAT CCCATACGATTGCAGCTGTATTAATCGATATGGATATTTCCAAGATTCAATCCAAATCAGAACAAGCTTAT GCAATTAGGAAAATCAAATCAGGCTTTCAACGTAGCTTGGATGACTATCATATTTATCAAATTGAAAGAAA ACAAACCTTTTCTTTTTCTCCGAAGCATCGCAGCCTCTCATCCACATCCCATTCCGAAGATTCTGATTTGG ATCTTTCTGAAGCAGCCGCCTTTTCAGGAAGTCTTACCTGCGAGTTTGTAAAAAAAAGCACTCAACATGCC AAGAATACCGTCACATGTTCCACAGCCGCTCATTCCCTATACACACTCAAAGAAGATGACAGCTCGAACCC CTCTGAAAAACGATTAGATAGTTGTTTCCGCAATTGGATTGAAAACAAACTAAGCGCCAATTCTCCAGATT CCTGGTCAGCGTTTATTCAAAAATTCGGAACACACTATATTGCATCAGCAACTTTTGGAGGGATAGGTTTC CAAGTGCTCAAACTATCTTTTGAACAGGTGGAGGATCTACATAGCAAAAAGATCTCCTTAGAAACCGCAGC AGCCAACTCTCTATTAAAAGGTTCTGTATCCAGCAGCACAGAATCTGGATACTCCAGCTATAGCTCCACGT CTTCTICTCATACGGTATTTTTAGGAGGAACGGTCTTACCTTCGGTTCATGATGAACGTTTAGACTTTAAA GATTGGTCGGAAAGTGTGCACCTGGAACCTGTTCCTATCCAGGTTTCTTTACAACCTATAACGAATTTACT AGTTCCTCTCCATTTTCCTAATATCGGTGCTGCAGAGCTCTCTAATAAACGAGAATCTCTTCAACAAGCGA TTCGAGTCTATCTCAAAGAACATAAAGTAGATGAGCAAGGAGAACGTACTACATTTACATCAGGAATCGAT AATCCTTCTTCCTGGTTTACCTTAGAAGCTGCCCACTCTCCTCTTATAGTCAGTACTCCTTACATTGCTTC GTGGTCTACGCTTCCTTATTTGTTCCCAACATTAAGAGAACGTTCTTCGGCAACCCCTATCGTTTTCTATT TTTGTGTAGATAATAATGAACATGCTTCGCAAAAAATATTAAACCAATCGTATTGCTTCCTCGGGTCCTTG CCTATTCGACAAAAAATTTTTGGTAGCGAATTTGCTAGTTTCCCCTATCTATCTTTCTATGGAAATGCAAA AGAGGCGTACTTTGATAACACGTACTACCCAACGCGTTGTGGGTGGATTGTTGAAAAGTTAAATACTACAC AAGATCAATTCCTCCGGGATGGAGACGAGGTGCGACTAAAACATGTTTCCAGCGGAAAGTATCTAGCAACA ACTCCTCTTAAGGATACCCATGGTACACTCACGCGTACAACGAACTGTGAAGATGCTATCTTTATTATTAA AAAATCTTCAGGTTATTGA SEQIDNO:6-CT153proteinsequence MTKPSFLYVIQPFSVFNPRLGRFSTDSDTYIEEENRLASFIESLPLEIFDIPSFMETAISNSPYILSWETT KDGALFTILEPKLSACAATCLVAPSIQMKSDAELLEEIKQALLRSSHDGVKYRITRESFSPEKKTPKVALV DDDIELIRNVDFLGRAVDIVKLDPINILNTVSEENILDYSFTRETAQLSADGRFGIPPGTKLFPKPSFDVE ISTSIFEETTSFTRSFSASVTFSVPDLAATMPLQSPPMVENGQKEICVIQKHLFPSYSPKLVDIVKRYKRE AKILINKLAFGMLWRHRAKSQILTEGSVRLDLQGFTESKYNYQIQVGSHTIAAVLIDMDISKIQSKSEQAY AIRKIKSGFQRSLDDYHIYQIERKQTFSFSPKHRSLSSTSHSEDSDLDLSEAAAFSGSLTCEFVKKSTQHA KNTVTCSTAAHSLYTLKEDDSSNPSEKRLDSCFRNWIENKLSANSPDSWSAFIQKFGTHYIASATFGGIGF QVLKLSFEQVEDLHSKKISLETAAANSLLKGSVSSSTESGYSSYSSTSSSHTVFLGGTVLPSVHDERLDFK DWSESVHLEPVPIQVSLQPITNLLVPLHFPNIGAAELSNKRESLQQAIRVYLKEHKVDEQGERTTFTSGID NPSSWFTLEAAHSPLIVSTPYIASWSTLPYLFPTLRERSSATPIVFYFCVDNNEHASQKILNQSYCFLGSL PIRQKIFGSEFASFPYLSFYGNAKEAYFDNTYYPTRCGWIVEKLNTTQDQFLRDGDEVRLKHVSSGKYLAT TPLKDTHGTLTRTTNCEDAIFIIKKSSGY SEQIDNO:7-CT601nucleotidesequence ATGCTCGCTAATCGCTTATTCTTAATAACCCTTTTAGGGTTAAGTTCGTCTGTTTACGGCGCAGGTAAAGC ACCGTCTTTGCAGGCTATTCTAGCCGAAGTCGAAGACACCTCCTCTCGTCTACACGCTCATCACAATGAGC TTGCTATGATCTCTGAACGCCTCGATGAGCAAGACACGAAACTACAGCAACTTTCGTCAACACAAGATCAT AACCTACCTCGACAAGTTCAGCGACTAGAAACGGACCAAAAAGCTTTGGCAAAAACACTGGCGATTCTTTC GCAATCCGTCCAAGATATTCGGTCTTCTGTACAAAATAAATTACAAGAAATCCAACAAGAACAAAAAAAAT TAGCACAAAATTTGCGAGCGCTTCGTAACTCTTTACAAGCTCTCGTTGATGGCTCTTCTCCAGAAAATTAT ATTGATTTCCTAACTGGTGAAACCCCGGAACATATTCATATTGTTAAACAAGGAGAGACCCTGAGCAAGAT CGCGAGTAAATATAACATCCCCGTCGTAGAATTAAAAAAACTTAATAAACTAAATTCGGATACTATTTTTA CAGATCAAAGAATTCGCCTTCCGAAAAAGAAATAG SEQIDNO:8-CT601proteinsequence MLANRLFLITLLGLSSSVYGAGKAPSLQAILAEVEDTSSRLHAHHNELAMISERLDEQDTKLQQLSSTQDH NLPRQVQRLETDQKALAKTLAILSQSVQDIRSSVQNKLQEIQQEQKKLAQNLRALRNSLQALVDGSSPENY IDFLTGETPEHIHIVKQGETLSKIASKYNIPVVELKKLNKLNSDTIFTDQRIRLPKKK SEQIDNO:9-CT279nucleotidesequence ATGGCATCCAAGTCTCGCCATTATCTTAATCAGCCTTGGTACATTATCTTATTCATCTTTGTTCTTAGTTT AATTGCTGGTACCCTCCTGTCTTCTGTGTATTATGTCCTTGCACCTATCCAACAGCAAGCTGCGGAATTCG ATCGCAATCAACAAATGCTAATGGCTGCACAAGTAATTTCTTCCGATAACACATTCCAAGTCTATGAAAAG GGAGATTGGCACCCAGCCCTATATAATACTAAAAAGCAGTTGCTAGAGATCTCCTCTACTCCTCCTAAAGT AACCGTGACAACTTTAAGCTCATATTTTCAAAACTTTGTTAGAGTCTTGCTTACAGATACACAAGGAAATC TTTCTTCATTCGAAGACCATAATCTCAATCTAGAAGAATTTTTATCTCAACCAACTCCTGTAATACATGGT CTTGCCCTTTATGTGGTCTACGCTATCCTACACAACGATGCAGCTTCCTCTAAATTATCTGCTTCCCAAGT AGCGAAAAATCCAACAGCTATAGAATCTATAGTTCTTCCTATAGAAGGTTTTGGTTTGTGGGGACCTATCT ATGGATTCCTTGCTCTAGAAAAAGACGGGAATACTGTTCTTGGTACTTCTTGGTATCAACATGGCGAGACT CCTGGATTAGGAGCAAATATCGCTAACCCTCAATGGCAAAAAAATTTCAGAGGCAAAAAAGTATTTCTAGT CTCAGCTTCTGGAGAAACAGATTTTGCTAAGACAACCCTAGGACTGGAAGTTATAAAAGGATCTGTATCTG CAGCATTAGGAGACTCACCTAAAGCTGCTTCTTCCATCGACGGAATTTCAGGAGCTACTTTGACTTGTAAT GGTGTTACCGAATCCTTCTCTCATTCTCTAGCTCCCTACCGCGCTTTGTTGACTTTCTTCGCCAACTCTAA ACCTAGTGGAGAGTCTCATGACCACTAA SEQIDNO:10-CT279proteinsequence MASKSRHYLNQPWYIILFIFVLSLIAGTLLSSVYYVLAPIQQQAAEFDRNQQMLMAAQVISSDNIFQVYEK GDWHPALYNTKKQLLEISSTPPKVTVTTLSSYFQNFVRVLLTDTQGNLSSFEDHNLNLEEFLSQPTPVIHG LALYVVYAILHNDAASSKLSASQVAKNPTAIESIVLPIEGFGLWGPIYGFLALEKDGNTVLGTSWYQHGET PGLGANIANPQWQKNFRGKKVFLVSASGETDFAKTTLGLEVIKGSVSAALGDSPKAASSIDGISGATLTCN GVTESFSHSLAPYRALLTFFANSKPSGESHDH SEQIDNO:11-CT443nucleotidesequence ATGCGAATAGGAGATCCTATGAACAAACTCATCAGACGAGCAGTGACGATCTTCGCGGTGACTAGTGTGGC GAGTTTATTTGCTAGCGGGGTGTTAGAGACCTCTATGGCAGAGTCTCTCTCTACAAACGTTATTAGCTTAG CTGACACCAAAGCGAAAGACAACACTTCTCATAAAAGCAAAAAAGCAAGAAAAAACCACAGCAAAGAGACT CCCGTAGACCGTAAAGAGGTTGCTCCGGTTCATGAGTCTAAAGCTACAGGACCTAAACAGGATTCTTGCTT TGGCAGAATGTATACAGTCAAAGTTAATGATGATCGCAATGTTGAAATCACACAAGCTGTTCCTGAATATG CTACGGTAGGATCTCCCTATCCTATTGAAATTACTGCTACAGGTAAAAGGGATTGTGTTGATGTTATCATT ACTCAGCAATTACCATGTGAAGCAGAGTTCGTACGCAGTGATCCAGCGACAACTCCTACTGCTGATGGTAA GCTAGTTTGGAAAATTGACCGCTTAGGACAAGGCGAAAAGAGTAAAATTACTGTATGGGTAAAACCTCTTA AAGAAGGTTGCTGCTTTACAGCTGCAACAGTATGCGCTTGTCCAGAGATCCGTTCGGTTACAAAATGTGGA CAACCTGCTATCTGTGTTAAACAAGAAGGCCCAGAGAATGCTTGTTTGCGTTGCCCAGTAGTTTACAAAAT TAATATAGTGAACCAAGGAACAGCAACAGCTCGTAACGTTGTTGTTGAAAATCCTGTTCCAGATGGTTACG CTCATTCTTCTGGACAGCGTGTACTGACGTTTACTCTTGGAGATATGCAACCTGGAGAGCACAGAACAATT ACTGTAGAGTTTTGTCCGCTTAAACGTGGTCGTGCTACCAATATAGCAACGGTTTCTTACTGTGGAGGACA TAAAAATACAGCAAGCGTAACAACTGTGATCAACGAGCCTTGCGTACAAGTAAGTATTGCAGGAGCAGATT GGTCTTATGTTTGTAAGCCTGTAGAATATGTGATCTCCGTTTCCAATCCTGGAGATCTTGTGTTGCGAGAT GTCGTCGTTGAAGACACTCTTTCTCCCGGAGTCACAGTTCTTGAAGCTGCAGGAGCTCAAATTTCTTGTAA TAAAGTAGTTTGGACTGTGAAAGAACTGAATCCTGGAGAGTCTCTACAGTATAAAGTTCTAGTAAGAGCAC AAACTCCTGGACAATTCACAAATAATGTTGTTGTGAAGAGCTGCTCTGACTGTGGTACTTGTACTTCTTGC GCAGAAGCGACAACTTACTGGAAAGGAGTTGCTGCTACTCATATGTGCGTAGTAGATACTTGTGACCCTGT TTGTGTAGGAGAAAATACTGTTTACCGTATTTGTGTCACCAACAGAGGTTCTGCAGAAGATACAAATGTTT CTTTAATGCTTAAATTCTCTAAAGAACTGCAACCTGTATCCTTCTCTGGACCAACTAAAGGAACGATTACA GGCAATACAGTAGTATTCGATTCGTTACCTAGATTAGGTTCTAAAGAAACTGTAGAGTTTTCTGTAACATT GAAAGCAGTATCAGCTGGAGATGCTCGTGGGGAAGCGATTCTTTCTTCCGATACATTGACTGTTCCAGTTT CTGATACAGAGAATACACACATCTATTAA SEQIDNO:12-CT443proteinsequence MRIGDPMNKLIRRAVTIFAVTSVASLFASGVLETSMAESLSTNVISLADTKAKDNTSHKSKKARKNHSKET PVDRKEVAPVHESKATGPKQDSCFGRMYTVKVNDDRNVEITQAVPEYATVGSPYPIEITATGKRDCVDVII TQQLPCEAEFVRSDPATTPTADGKLVWKIDRLGQGEKSKITVWVKPLKEGCCFTAATVCACPEIRSVTKCG QPAICVKQEGPENACLRCPVVYKINIVNQGTATARNVVVENPVPDGYAHSSGQRVLTFTLGDMQPGEHRTI TVEFCPLKRGRATNIATVSYCGGHKNTASVTTVINEPCVQVSIAGADWSYVCKPVEYVISVSNPGDLVLRD VVVEDTLSPGVTVLEAAGAQISCNKVVWTVKELNPGESLQYKVLVRAQTPGQFTNNVVVKSCSDCGTCTSC AEATTYWKGVAATHMCVVDTCDPVCVGENTVYRICVTNRGSAEDTNVSLMLKFSKELQPVSFSGPTKGTIT GNTVVFDSLPRLGSKETVEFSVTLKAVSAGDARGEAILSSDTLTVPVSDTENTHIY SEQIDNO:13-CT372nucleotidesequence ATGCAGGCTGCACACCATCACTATCACCGCTACACAGATAAACTGCACAGACAAAACCATAAAAAAGATCT CATCTCTCCCAAACCTACCGAACAAGAGGCGTGCAATACTTCTTCCCTTAGTAAGGAATTAATCCCTCTAT CAGAACAAAGAGGCCTTTTATCCCCCATCTGTGACTTTATTTCGGAACGCCCTTGCTTACACGGAGTTTCT GTTAGAAATCTCAAGCAAGCGCTAAAAAATTCTGCAGGAACCCAAATTGCACTGGATTGGTCTATTCTCCC TCAATGGTTCAATCCTCGGGTCTCTCATGCCCCTAAGCTTTCTATCCGAGACTTTGGGTATAGCGCACACC AAACTGTTACCGAAGCCACTCCTCCTTGCTGGCAAAACTGCTTTAATCCATCTGCGGCCGTTACTATCTAT GATTCCTCATATGGGAAAGGGGTCTTTCAAATATCCTATACCCTTGTCCGCTATTGGAGAGAGAATGCTGC GACTGCTGGCGATGCTATGATGCTCGCAGGGAGTATCAATGATTATCCCTCTCGTCAGAACATTTTCTCTC AGTTTACTTTCTCCCAAAACTTCCCAAATGAACGGGTGAGTCTGACAATTGGTCAGTACTCACTCTATGCA ATAGACGGAACATTATACAATAACGATCAACAACTTGGATTCATTAGTTACGCATTATCACAAAATCCAAC AGCAACTTATTCCTCTGGAAGTCTTGGAGCTTACCTACAAGTCGCTCCTACCGCAAGCACAAGTCTTCAAA TAGGATTTCAAGACGCTTATAATATCTCCGGATCCTCTATCAAATGGAGTAACCTTACAAAAAATAGATAC AATTTTCACGGTTTTGCTTCCTGGGCTCCCCGCTGTTGCTTAGGATCTGGCCAGTACTCCGTGCTTCTTTA TGTGACTAGACAAGTTCCAGAACAGATGGAACAAACAATGGGATGGTCAGTCAATGCGAGTCAACACATAT CTTCTAAACTGTATGTGTTTGGAAGATACAGCGGTGTTACAGGACATGTGTTCCCGATTAACCGCACGTAT TCATTCGGTATGGCCTCTGCAAATTTATTTAACCGTAACCCACAAGATTTATTTGGAATTGCTTGCGCATT CAATAATGTACACCTCTCTGCTTCTCCAAATACTAAAAGAAAATACGAAACTGTAATCGAAGGGTTTGCAA CTATCGGTTGCGGCCCCTATCTTTCTTTCGCTCCAGACTTCCAACTCTACCTCTACCCAGCTCTTCGTCCA AACAAACAATCTGCCCGTGTTTATAGCGTGCGAGCTAATTTAGCTATCTAA SEQIDNO:14-CT372proteinsequence MQAAHHHYHRYTDKLHRQNHKKDLISPKPTEQEACNTSSLSKELIPLSEQRGLLSPICDFISERPCLHGVS VRNLEQALKNSAGTQIALDWSILPQWFNPRVSHAPKLSIRDFGYSAHQTVTEATPPCWQNCFNPSAAVTIY DSSYGKGVFQISYTLVRYWRENAATAGDAMMLAGSINDYPSRQNIFSQFTFSQNFPNERVSLTIGQYSLYA IDGTLYNNDQQLGFISYALSQNPTATYSSGSLGAYLQVAPTASTSLQIGFQDAYNISGSSIKWSNLTKNRY NFHGFASWAPRCCLGSGQYSVLLYVTRQVPEQMEQTMGWSVNASQHISSKLYVFGRYSGVTGHVFPINRTY SFGMASANLFNRNPQDLFGIACAFNNVHLSASPNTKRKYETVIEGFATIGCGPYLSFAPDFQLYLYPALRP NKQSARVYSVRANLAI SEQIDNO:15:CT043nucleotidesequence ATGTCCAGGCAGAATGCTGAGGAAAATCTAAAAAATTTTGCTAAAGAGCTTAAACTCCCCGACGTGGCCTT CGATCAGAATAATACGTGCATTTTGTTTGTTGATGGAGAGTTTTCTCTTCACCTGACCTACGAAGAACACT CTGATCGCCTTTATGTTTACGCACCTCTTCTTGACGGACTGCCAGACAATCCGCAAAGAAGGTTAGCTCTA TATGAGAAGTTGTTAGAAGGCTCTATGCTCGGAGGCCAAATGGCTGGTGGAGGGGTAGGAGTCGCTACTAA GGAACAGTTGATCTTAATGCACTGCGTGTTAGACATGAAGTATGCAGAGACCAACCTACTCAAAGCTTTTG CACAGCTTTTTATTGAAACCGTTGTGAAATGGCGAACTGTTTGTTCTGATATCAGCGCTGGACGAGAACCC ACTGTTGATACCATGCCACAAATGCCTCAAGGGGGTGGCGGAGGAATTCAACCTCCTCCAGCAGGAATCCG TGCATAA SEQIDNO:16:CT043proteinsequence MSRQNAEENLKNFAKELKLPDVAFDQNNTCILFVDGEFSLHLTYEEHSDRLYVYAPLLDGLPDNPQRRLAL YEKLLEGSMLGGQMAGGGVGVATKEQLILMHCVLDMKYAETNLLKAFAQLFIETVVKWRTVCSDISAGREP TVDTMPQMPQGGGGGIQPPPAGIRA SEQIDNO:17-CT681proteinsequence MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPCATWCDAISMRVGYYGDFVFD RVLKTDVNKEFQMGAKPTTDTGNSAAPSTLTARENPAYGRHMQDAEMFTNAACMALNIWDRFDVFCTLGAT SGYLKGNSASFNLVGLFGDNENQKTVKAESVPNMSFDQSVVELYTDTTFAWSVGARAALWECGCATLGASF QYAQSKPKVEELNVLCNAAEFTINKPKGYVGKEFPLDLTAGTDAATGTKDASIDYHEWQASLALSYRLNMF TPYIGVKWSRASFDADTIRIAQPKSATAIFDTTTLNPTIAGAGDVKTGAEGQLGDTMQIVSLQLNKMKSRK SCGIAVGTTIVDADKYAVTVETRLIDERAAHVNAQFRF SEQIDNO:18-CT681nucleotidesequence ATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTTGAGTTCTGCTTCCTCCTTGCAAGCTCTGCC TGTGGGGAATCCTGCTGAACCAAGCCTTATGATCGACGGAATTCTGTGGGAAGGTTTCGGCGGAGATCCTT GCGATCCTTGCGCCACTTGGTGTGACGCTATCAGCATGCGTGTTGGTTACTACGGAGACTTTGTTTTCGAC CGTGTTTTGAAAACAGATGTGAATAAAGAATTTCAGATGGGTGCCAAGCCTACAACTGATACAGGCAATAG TGCAGCTCCATCCACTCTTACAGCAAGAGAGAATCCTGCTTACGGCCGACATATGCAGGATGCTGAGATGT TTACAAATGCCGCTTGCATGGCATTGAATATTTGGGATCGTTTTGATGTATTCTGTACATTAGGAGCCACC AGTGGATATCTTAAAGGAAACTCTGCTTCTTTCAATTTAGTTGGATTGTTTGGAGATAATGAAAATCAAAA AACGGTCAAAGCGGAGTCTGTACCAAATATGAGCTTTGATCAATCTGTTGTTGAGTTGTATACAGATACTA CTTTTGCGTGGAGCGTCGGCGCTCGCGCAGCTTTGTGGGAATGTGGATGTGCAACTTTAGGAGCTTCATTC CAATATGCTCAATCTAAACCTAAAGTAGAAGAATTAAACGTTCTCTGCAATGCAGCAGAGTTTACTATTAA TAAACCTAAAGGGTATGTAGGTAAGGAGTTTCCTCTTGATCTTACAGCAGGAACAGATGCTGCGACAGGAA CTAAGGATGCCTCTATTGATTACCATGAATGGCAAGCAAGTTTAGCTCTCTCTTACAGACTGAATATGTTC ACTCCCTACATTGGAGTTAAATGGTCTCGAGCAAGCTTTGATGCCGATACGATTCGTATAGCCCAGCCAAA ATCAGCTACAGCTATTTTTGATACTACCACGCTTAACCCAACTATTGCTGGAGCTGGCGATGTGAAAACTG GCGCAGAGGGTCAGCTCGGAGACACAATGCAAATCGTTTCCTTGCAATTGAACAAGATGAAATCTAGAAAA TCTTGCGGTATTGCAGTAGGAACAACTATTGTGGATGCAGACAAATACGCAGTTACAGTTGAGACTCGCTT GATCGATGAGAGAGCAGC SEQIDNO:19-TC0210proteinsequence MMKRLLCVLLSTSVFSSPMLGYSAPKKDSSTGICLAASQSDRELSQEDLLKEVSRGFSKVAAQATPGVVYI ENFPKTGSQAIASPGNKRGFQENPFDYFNDEFFNRFFGLPSHREQPRPQQRDAVRGTGFIVSEDGYVVTNH HVVEDAGKIHVTLHDGQKYTAKIIGLDPKTDLAVIKIQAKNLPFLTFGNSDQLQIGDWSIAIGNPFGLQAT VTVGVISAKGRNQLHIVDFEDFIQTDAAINPGNSGGPLLNIDGQVIGVNTAIVSGSGGYIGIGFAIPSLMA KRVIDQLISDGQVTRGFLGVTLQPIDSELAACYKLEKVYGALITDVVKGSPAEKAGLRQEDVIVAYNGKEV ESLSALRNAISLMMPGTRVVLKVVREGKFIEIPVTVTQIPAEDGVSALQKMGVRVQNLTPEICKKLGLASD TRGIFVVSVEAGSPAASAGVVPGQLILAVNRQRVSSVEELNQVLKNAKGENVLLMVSQGEVIRFVVLKSDE SEQIDNO:20-TC0052proteinsequence MKKLLKSVLAFAVLGSASSLHALPVGNPAEPSLMIDGILWEGFGGDPCDPCTTWCDAISLRLGYYGDFVFD RVLKTDVNKQFEMGAAPTGDADLTTAPTPASRENPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATS GYLKGNSAAFNLVGLFGRDETAVAADDIPNVSLSQAVVELYTDTAFAWSVGARAALWECGCATLGASFQYA QSKPKVEELNVLCNAAEFTINKPKGYVGQEFPLNIKAGTVSATDTKDASIDYHEWQASLALSYRLNMFTPY IGVKWSRASFDADTIRIAQPKLETSILKMTTWNPTISGSGIDVDTKITDTLQIVSLQLNKMKSRKSCGLAI GTTIVDADKYAVTVETRLIDERAAHVNAQFRF SEQIDNO:21-TC0052nucleotidesequence ATGAAAAAACTCTTGAAATCGGTATTAGCATTTGCCGTTTTGGGTTCTGCTTCCTCCTTGCATGCTCTGCC TGTGGGGAATCCTGCTGAACCAAGCCTTATGATTGACGGGATTCTTTGGGAAGGTTTCGGTGGAGATCCTT GCGATCCTTGCACAACTTGGTGTGATGCCATCAGCCTACGTCTCGGCTACTATGGGGACTTCGTTTTTGAT CGTGTTTTGAAAACAGACGTGAACAAACAGTTCGAAATGGGAGCAGCTCCTACAGGAGATGCAGACCTTAC TACAGCACCTACTCCTGCATCAAGAGAGAATCCCGCTTATGGCAAGCATATGCAAGATGCAGAAATGTTCA CTAATGCTGCGTACATGGCTTTAAACATTTGGGACCGTTTCGATGTATTTTGTACATTGGGAGCAACTAGC GGATATCTTAAAGGTAATTCTGCCGCCTTTAACTTAGTTGGTCTGTTTGGAAGAGATGAAACTGCAGTTGC AGCTGACGACATACCTAACGTCAGCTTGTCTCAAGCTGTTGTCGAACTCTACACAGACACAGCTTTCGCTT GGAGCGTCGGTGCTAGAGCAGCTTTATGGGAGTGCGGATGTGCAACTTTAGGAGCTTCCTTCCAATATGCT CAATCTAAGCCAAAAGTAGAGGAATTAAACGTTCTCTGTAATGCGGCAGAATTCACTATTAACAAGCCTAA AGGATACGTTGGACAAGAGTTTCCTCTTAACATTAAAGCTGGAACAGTTAGCGCTACAGATACTAAAGATG CTTCCATCGATTACCATGAGTGGCAAGCAAGCTTGGCTTTGTCTTACAGACTGAATATGTTCACTCCTTAC ATTGGAGTTAAGTGGTCTAGAGCAAGCTTTGATGCCGACACTATCCGCATTGCGCAGCCTAAGCTTGAGAC CTCTATCTTAAAAATGACCACTTGGAACCCAACGATCTCTGGATCTGGTATAGACGTTGATACAAAAATCA CGGATACATTACAAATTGTTTCCTTGCAGCTCAACAAGATGAAATCCAGAAAATCTTGCGGTCTTGCAATT GGAACAACAATTGTAGATGCTGATAAATATGCAGTTACTGTTGAGACACGCTTGATCGATGAAAGAGCAGC TCACGTAAATGCTCAGTTCCGTTTCTAA SEQIDNO:22-TC0106proteinsequence MLTNFTFRNCLLFFVTLSSVPVFSAPQPRVTLPSGANKIGSEAWIEQKVRQYPELLWLVEPSPAGTSLNAP SGMIFSPLLFQKKVPAFDIAVRSLIHLHLLIQGSRQAYAQLVQLQANESPMTFKQFLTLHKQLSLFLNSPK EFYDSVKILETAIILRHLGCSTKAVATFKPYFSETQKEVFYTKALHVLHTFPELSPSFARLSPEQKTLFFS LRKLANYDELLSLTNAPSLQLLSAVRSRRALLALDLYLYALDFCGEQGISSQFHMDFSPLQSMLQQYATVE EAFSRYFTYRANRLGFAGSSRTEMALVRIATLMNLSPSEAAILTTSFKSLSLEDAESLVNSFYTNKGDSLA LSLRGLPTLISELTRAAHGNTNAEARAQQIYATTLSLVAKSLKAHKEMQNKQILPEEVVLDFSETASSCQG LDIFSENVAVQIHLNGSVSIHL SEQIDNO:23:CMhomologofCT601= TC_0551 ATGGCATCCAAGTCTCGTCATTATCTTAACCAGCCTTGGTACATTATCTTATTCATCTTTGTTCTTAGTCT GGTTGCTGGTACCCTTCTTTCTTCAGTTTCCTATGTTCTATCTCCAATCCAAAAACAAGCTGCAGAATTTG ATCGTAATCAGCAAATGTTGATGGCCGCACAAATTATTTCCTATGACAATAAATTCCAAATATATGCTGAA GGGGATTGGCAACCTGCTGTCTATAATACAAAAAAACAGATACTAGAAAAAAGCTCTTCCACTCCACCACA AGTGACTGTGGCGACTCTATGCTCTTATTTTCAAAATTTTGTTAGAGTTTTGCTTACAGACTCCCAAGGGA ATCTTTCTTCTTTTGAAGATCACAATCTTAACCTAGAAGAGTTCTTATCCCACCCCACATCTTCAGTACAA GATCACTCTCTGCATGTAATTTATGCTATTCTAGCAAACGATGAATCCTCTAAAAAGTTATCATCCTCCCA AGTAGCAAAAAATCCGGTATCCATAGAGTCTATTATTCTTCCTATAAAAGGATTTGGTTTATGGGGACCAA TCTATGGATTTCTTGCTTTAGAAAAGGACGGTAATACGGTTCTAGGGACATGCTGGTATCAACATGGTGAG ACTCCAGGATTAGGAGCAAATATAACTAATCCCCAATGGCAACAAAATTTCAGAGGAAAAAAAGTATTTCT CGCTTCCTCTTCCGGAGAAACCGATTTTGCTAAAACAACTCTAGGACTAGAAGTTATAAAAGGATCTGTTT CTGCATTATTAGGGGACTCTCCCAAAGCTAATTCCGCTGTTGATGGAATTTCAGGAGCTACACTGACCTGT AATGGAGTTACTGAAGCTTTTGCTAATTCGCTAGCTCCTTACCGCCCCTTATTGACTTTCTTCGCCAATCT TAACTCTAGTGGAGAATCTCATGACAACCAATAA SEQIDNO:24:CMhomologueofCT601proteinsequence= TC_0551protein sequence MASKSRHYLNQPWYIILFIFVLSLVAGTLLSSVSYVLSPIQKQAAEFDRNQQMLMAAQIISYDNKFQIYAE GDWQPAVYNTKKQILEKSSSTPPQVTVATLCSyFQNFvRVLLTDSQGNLSSFEDHNLNLEEFLSHPTSSVQ DHSLHVIYAILANDESSKKLSSSQVAKNPVSIESIILPIKGFGLWGPIYGFLALEKDGNTVLGTCWYQHGE TPGLGANITNPQWQQNFRGKKVFLASSSGETDFAKTTLGLEVIKGSVSALLGDSPKANSAVDGISGATLTC NGVTEAFANSLAPYRPLLTFFANLNSSGESHDNQ SEQIDNO:25:CMhomologueofCT372= TC_0651nucleotidesequence ATGAATGGAAAAGTTCTGTGTGAGGTTTCTGTGTCCTTCCGTTCGATTCTGCTGACGGCTCTGCTTTCACT TTCTTTTACAAACACTATGCAGGCTGCACACCATCATTATCACCGTTATGATGATAAACTACGCAGACAAT ACCATAAAAAGGACTTGCCCACTCAAGAGAATGTTCGGAAAGAGTTTTGTAATCCCTACTCTCATAGTAGT GATCCTATCCCTTTGTCACAACAACGAGGAGTCCTATCTCCTATCTGTGATTTAGTCTCAGAGTGCTCGTT TTTGAACGGGATTTCCGTTAGGAGTCTTAAACAAACACTGAAAAATTCTGCTGGGACTCAAGTTGCTTTAG ACTGGTCTATCCTTCCTCAATGGTTCAATCCTAGATCCTCTTGGGCTCCTAAGCTCTCTATTCGAGATCTT GGATATGGTAAACCCCAGTCCCTTATTGAAGCAGATTCCCCTTGTTGTCAAACCTGCTTCAACCCATCTGC TGCTATTACGATTTACGATTCTTCATGTGGGAAGGGTGTTGTCCAAGTGTCATACACCCTTGTTCGTTATT GGAGAGAAACGGCTGCACTTGCAGGGCAAACTATGATGCTTGCAGGAAGTATTAATGATTATCCTGCTCGC CAAAACATATTCTCTCAACTTACATTTTCCCAAACTTTCCCTAATGAGAGAGTAAATCTAACTGTTGGTCA ATACTCTCTTTACTCGATAGACGGAACGCTGTACAACAATGATCAGCAGCTAGGATTTATTAGTTATGCGT TGTCGCAAAATCCAACAGCGACTTATTCCTCTGGAAGCCTTGGCGCCTATCTACAAGTCGCTCCAACAGAA AGCACCTGTCTTCAAGTTGGGTTCCAAGATGCCTATAATATTTCAGGTTCCTCGATCAAATGGAATAATCT TACAAAAAATAAGTATAACTTCCATGGCTATGCATCTTGGGCTCCACACTGTTGCTTAGGACCTGGACAAT ACTCTGTTCTTCTTTATGTAACCAGAAAGGTTCCTGAGCAAATGATGCAGACAATGGGCTGGTCTGTGAAT GCAAGTCAATACATCTCTTCTAAACTTTATGTATTIGGAAGATACAGCGGAGTCACAGGCCAATTGTCTCC TATTAACCGAACCTATTCATTTGGCTTAGTCTCTCCTAATTTATTGAACCGTAACCCACAAGACTTATTTG GAGTAGCTTGCGCATTCAATAATATACACGCCTCCGCCTTTCAAAATGCTCAAAGAAAATATGAAACTGTG ATCGAGGGATTTGCAACTATTGGTTGCGGACCTTACATCTCCTTTGCTCCAGATTTCCAACTTTACCTCTA TCCTGCTCTGCGTCCAAATAAACAAAGCGCCCGAGTCTATAGCGTTCGCGCAAACCTAGCTATTTAG SEQIDNO:26:CMhomologueofCT372= TC_0651proteinsequence MNGKVLCEVSVSFRSILLTALLSLSFTNTMQAAHHHYHRYDDKLRRQYHKKDLPTQENVRKEFCNPYSHSS DPIPLSQQRGVLSPICDLVSECSFLNGISVRSLKQTLKNSAGTQVALDWSILPQWFNPRSSWAPKLSIRDL GYGKPQSLIEADSPCCQTCFNPSAAITIYDSSCGKGVVQVSYTLVRYWRETAALAGQTMMLAGSINDYPAR QNIFSQLTFSQTFPNERVNLTVGQYSLYSIDGTLYNNDQQLGFISYALSQNPTATYSSGSLGAYLQVAPTE STCLQVGFQDAYNISGSSIKWNNLTKNKYNFHGYASWAPHCCLGPGQYSVLLYVTRKVPEQMMQTMGWSVN ASQYISSKLYVFGRYSGVTGQLSPINRTYSFGLVSPNLLNRNPQDLFGVACAFNNIHASAFQNAQRKYETV IEGFATIGCGPYISFAPDFQLYLYPALRPNKQSARVYSVRANLAI SEQIDNO:27:CMhomologueofCT443= TC_0727 ATGCGAATAGGAGATCCTATGAACAAACTCATCAGACGAGCTGTGACGATCTTCGCGGTGACTAGTGTGGC GAGTTTATTTGCTAGCGGGGTGTTAGAGACCTCTATGGCAGAGTCTCTCTCTACCAACGTTATTAGCTTAG CTGACACCAAAGCGAAAGAGACCACTTCTCATCAAAAAGACAGAAAAGCAAGAAAAAATCATCAAAATAGG ACTTCCGTAGTCCGTAAAGAGGTTACTGCAGTTCGTGATACTAAAGCTGTAGAGCCTAGACAGGATTCTTG CTTTGGCAAAATGTATACAGTCAAAGTTAATGATGATCGTAATGTAGAAATCGTGCAGTCCGTTCCTGAAT ATGCTACGGTAGGATCTCCATATCCTATTGAGATTACTGCTATAGGGAAAAGAGACTGTGTTGATGTAATC ATTACACAGCAATTACCATGCGAAGCAGAGTTTGTTAGCAGTGATCCAGCTACTACTCCTACTGCTGATGG TAAGCTAGTTTGGAAAATTGATCGGTTAGGACAGGGCGAAAAGAGTAAAATTACTGTATGGGTAAAACCTC TTAAAGAAGGTTGCTGCTITACAGCTGCAACGGTTTGTGCTTGTCCAGAGATCCGTTCGGTTACGAAATGT GGCCAGCCTGCTATCTGTGTTAAACAGGAAGGTCCAGAAAGCGCATGTTTGCGTTGCCCAGTAACTTATAG AATTAATGTAGTCAACCAAGGAACAGCAACAGCACGTAATGTTGTTGTGGAAAATCCTGTTCCAGATGGCT ATGCTCATGCATCCGGACAGCGTGTATTGACATATACTCTTGGGGATATGCAACCTGGAGAACAGAGAACA ATCACCGTGGAGTTTTGTCCGCTTAAACGTGGTCGAGTCACAAATATTGCTACAGTTTCTTACTGTGGTGG ACACAAAAATACTGCTAGCGTAACAACAGTGATCAATGAGCCTTGCGTGCAAGTTAACATCGAGGGAGCAG ATTGGTCTTATGTTTGTAAGCCTGTAGAATATGTTATCTCTGTTTCTAACCCTGGTGACTTAGTTTTACGA GACGTTGTAATTGAAGATACGCTTTCTCCTGGAATAACTGTTGTTGAAGCAGCTGGAGCTCAGATTTCTTG TAATAAATTGGTTTGGACTTTGAAGGAACTCAATCCTGGAGAGTCTTTACAATATAAGGTTCTAGTAAGAG CTCAAACTCCAGGGCAATTCACAAACAACGTTGTTGTGAAAAGTTGCTCTGATTGCGGTATTTGTACTTCT TGCGCAGAAGCAACAACTTACTGGAAAGGAGTTGCTGCTACTCATATGTGCGTAGTAGATACTTGTGATCC TATTTGCGTAGGAGAGAACACTGTTTATCGTATCTGTGTGACAAACAGAGGTTCTGCTGAAGATACAAATG TGTCCTTAATTTTGAAATTCTCTAAAGAATTACAACCTATATCTTTCTCTGGACCAACTAAAGGAACCATT ACAGGAAACACGGTAGTGTTTGATTCGTTACCTAGATTAGGTTCTAAAGAAACTGTAGAGTTTTCTGTAAC GTTGAAAGCAGTATCCGCTGGAGATGCTCGTGGGGAAGCTATTCTTTCTTCCGATACATTGACAGTTCCTG TATCTGATACGGAGAATACACATATCTATTAA SEQIDNO:28:CMhomologueofCT443= TC_0727 MRIGDPMNKLIRRAVTIFAVTSVASLFASGVLETSMAESLSTNVISLADTKAKETTSHQKDRKARKNHQNR TSVVRKEVTAVRDTKAVEPRQDSCFGKMYTVKVNDDRNVEIVQSVPEYATVGSPYPIEITAIGKRDCVDVI ITQQLPCEAEFVSSDPATTPTADGKLVWKIDRLGQGEKSKITVWVKPLKEGCCFTAATVCACPEIRSVTKC GQPAICVKQEGPESACLRCPVTYRINVVNQGTATARNVVVENPVPDGYAHASGQRVLTYTLGDMQPGEQRT ITVEFCPLKRGRVTNIATVSYCGGHKNTASVTTVINEPCVQVNIEGADWSYVCKPVEYVISVSNPGDLVLR DVVIEDTLSPGITVVEAAGAQISCNKLVWTLKELNPGESLQYKVLVRAQTPGQFTNNVVVKSCSDCGICTS CAEATTYWKGVAATHMCVVDTCDPICVGENTVYRICVTNRGSAEDTNVSLILKFSKELQPISFSGPTKGTI TGNTVVFDSLPRLGSKETVEFSVTLKAVSAGDARGEAILSSDTLTVPVSDTENTHTY SEQIDNO:29:CMhomologueofCT043= TC_0313nucleotidesequence ATGTCCAGACAGAATGCTGAGGAAAATCTAAAAAATTTTGCTAAAGAGCTCAAGCTCCCCGACGTGGCCTT CGATCAGAATAATACGTGCATTTTGTTTGTTGATGGAGAGTTTTCTCTTCACCTGACCTACGAAGAGCACT CTGATCGCCTTTATGTTTACGCACCTCTCCTTGACGGACTCCCAGATAATCCGCAAAGAAAGTTGGCTCTG TATGAGAAATTGTTGGAAGGCTCTATGCTCGGAGGCCAAATGGCTGGTGGAGGAGTAGGAGTTGCTACTAA AGAACAGTTGATCCTAATGCATTGCGTGTTAGATATGAAATATGCAGAGACTAATCTATTGAAAGCTTTTG CACAGCTTTTCATTGAAACTGTTGTGAAATGGCGAACGGTCTGTTCTGATATCAGCGCTGGACGAGAACCT TCCGTTGACACTATGCCTCAAATGCCTCAAGGAGGCAGCGGAGGAATTCAACCTCCTCCAACAGGAATTCG TGCGTAG SEQIDNO:30:CMhomologueofCT043= TC_0313proteinsequence MSRQNAEENLKNFAKELKLPDVAFDQNNTCILFVDGEFSLHLTYEEHSDRLYVYAPLLDGLPDNPQRKLAL YEKLLEGSMLGGQMAGGGVGVATKEQLILMHCVLDMKYAETNLLKAFAQLFIETVVKWRTVCSDISAGREP SVDTMPQMPQGGSGGIQPPPTGIRA SEQIDNO:31-TC0431proteinsequence MPHSPFLYVVQPHSVFNPRLGERHPITLDFIKEKNRLADFIENLPLEIFGAPSFLENASLEASYVLSREST KDGTLFTVLEPKLSACVATCLVDSSIPMEPDNELLEEIKHTLLKSSCDGVQYRVTRETLQNKDEAPRVSLV ADDIELIRNVDFLGRSVDIVKLDPLNIPNTVSEENALDYSFTRETAKLSPDGRVGIPQGTKILPAPSLEVE ISTSIFEETSSFEQNFSSSITFCVPPLTSFSPLQEPPLVGAGQQEILVTKKHLFPSYTPKLIDIVKRHKRD AKILVNKIQFEKLWRSHAKSQILKEGSVRLDLQGFTGELFNYQLQVGSHTIAAVLIDPEIANVKSLPEQTY AVRKIKSGFQCSLDDQHIYQVAVKKHLSLSSQPPKISPLSQSESSDLSLFEAAAFSASLTYEFVKKNTYHA KNTVTCSTVSHSLYILKEDDGANAAEKRLDNSFRNWVENKLNANSPDSCTAFIQKFGTHYITSATFGGSGF QVLKLSFEQVEGLRSKKISLEAAAANSLLKSSVSNSTESGYSTYDSSSSSHTVFLGGTVLPSVHDGQLDFK DWSESVCLEPVPIHISLLPLTDLLTPLYFPETDTTELSNKRNALQQAVRVYLKDHRSAKQSERSVFTAGIN SPSSWFTLESANSPLVVSSPYMTYWSTLPYLFPTLKERSSAAPIVFYFCVDNNEHASQKILNQTYCFIGSL PIRQKIFGREFAENPYLSFYGRFGEAYFDGGYPERCGWIVEKLNTTKDQILRDEDEVQLKHVYSGEYLSTI PIKDSHCTLSRTCTESNAVFIIKKPSSY SEQIDNO:32CMhomologueofCT279= TC0890nucleotidesequence ATGCTCGCTAATCGGTTATTTCTAATCACCCTTATAGGTTTTGGCTATTCTGCTTACGGTGCCAGCACAGG GAAATCACCTTCTTTACAGGTTATTTTAGCTGAAGTCGAGGATACATCTTCGCGCTTACAAGCTCATCAGA ATGAGCTTGITATGCTCTCGGAACGTTTAGATGAGCAAGACACAAAACTTCAACAACTCTCGTCAACTCAG GCCCGTAATCTTCCTCAACAAGTTCAACGGCTTGAGATTGATCTGAGAGCTCTGGCTAAAACAGCTGCTGT GCTCTCGCAATCTGTTCAGGATATCCGATCATCCGTGCAAAATAAATTACAAGAAATCCAACAAGAACAAA AAAATTTAGCTCAAAATTTACGAGCGCTTCGCAACTCCTTACAAGCACTAGTTGATGGCTCTTCCCCAGAA AATTATATTGATTTTTTGGCCGGGGAGACACCTGAACATATTCACGTTGTTAAACAAGGAGAAACCCTGAG TAAAATCGCTAGTAAGTACAATATCCCTGTCGCAGAATTGAAAAAACTTAATAAATTAAATTCCGATACTA TTTTTACTGATCAAAGAATCCGACTTCCAAAAAAGAAATAA SEQIDNO:33:CMhomologueofCT279= TC_0890proteinsequence MLANRLFLITLIGFGYSAYGASTGKSPSLQVILAEVEDTSSRLQAHQNELVMLSERLDEQDTKLQQLSSTQ ARNLPQQVQRLEIDLRALAKTAAVLSQSVQDIRSSVQNKLQEIQQEQKNLAQNLRALRNSLQALVDGSSPE NYIDFLAGETPEHIHVVKQGETLSKIASKYNIPVAELKKLNKLNSDTIFTDQRIRLPKKK SEQIDNO:34-TC0660proteinsequence MSMYIKRKKAWMTFLAIVCSFCLAGCSKESKDSVSEKFIVGTNATYPPFEFVDERGETVGFDIDLAREISK KLGKKLEVREFAFDALVLNLKQHRIDAIMAGVSITSSRLKEILMIPYYGEEIKSLVLVFKDGDSKSLPLDQ YNSVAVQTGTYQEEYLQSLPGVRIRSFDSTLEVLMEVLHSKSPIAVLEPSIAQVVLKDFPTLTTETIDLPE DKWVLGYGIGVASDRPSLASDIEAAVQEIKKEGVLAELEQKWGLNG SEQIDNO:35-TC0741proteinsequence MTTPISNSPSSIPTVTVSTTTASSGSLGTSTVSSTTTSTSVAQTATTTSSASTSIIQSSGENIQSTTGTPS PITSSVSTSAPSPKASATANKTSSAVSGKITSQETSEESETQATTSDGEVSSNYDDVDTPTNSSDSTVDSD YQDVETQYKTISNNGENTYETIGSHGEKNTHVQESHASGTGNPINNQQEAIRQLRSSTYTTSPRNENIFSP GPEGLPNMSLPSYSPTDKSSLLAFLSNPNTKAKMLEHSGHLVFIDTTRSSFIFVPNGNWDQVCSMKVQNGK TKEDLGLKDLEDMCAKFCTGYNKFSSDWGNRVDPLVSSKAGIESGGHLPSSVIINNKFRTCVAYGPWNPKE NGPNYTPSAWRRGHRVDFGKIFDGTAPFNKINWGSSPTPGDDGISFSNETIGSEPFATPPSSPSQTPVINV NVNVGGTNVNIGDTNVSKGSGTPTSSQSVDMSTDTSDLDTSDIDTNNQTNGDINTNDNSNNVDGSLSDVDS RVEDDDGVSDTESTNGNDSGKTTSTEENGDPSGPDILAAVRKHLDTVYPGENGGSTEGPLPANQNLGNVIH DVEQNGSAKETIITPGDTGPTDSSSSVDADADVEDTSDTDSGIGDDDGVSDTESTNGNNSGKTTSTEENGD PSGPDILAAVRKHLDTVYPGENGGSTEGPLPANQNLGNVIHDVEQNGAAQETIITPGDTESTDTSSSVNAN ADLEDVSDADSGFGDDDGISDTESTNGNDSGKNTPVGDGGTPSGPDILAAVRKHLDTVYPGENGGSTERPL PANQNLGDIIHDVEQNGSAKETVVSPYRGGGGNTSSPIGLASLLPATPSTPLMTTPRTNGKAAASSLMIKG GETQAKLVKNGGNIPGETTLAELLPRLRGHLDKVFTSDGKFTNLNGPQLGAIIDQFRKETGSGGIIAHTDS VPGENGTASPLTGSSGEKVSLYDAAKNVTQALTSVTNKVTLAMQGQKLEGIINNNNTPSSIGQNLFAAARA TTQSLSSLIGTVQ
REFERENCES
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