Anti-Mycoplasma spp. subunit vaccine

Abstract

Provided in the present invention are anti-Mycoplasma spp. subunit vaccines, especially proteins suitable for being used as the active ingredient of the Mycoplasma spp. subunit vaccines, and a vaccine prepared therefrom. Upon experimenting, it is confirmed that the proteins can elicit an immune response having sufficient strength to avoid the infection of Mycoplasma spp. in pigs. The vaccine can comprise one of the aforementioned proteins as an active ingredient, or can comprise two or more of the proteins to form a form of cocktail vaccine. The vaccine of the present invention is not only more safe than conventional vaccines, but also has equivalent or even better immune effects.

Claims

1. A composition for preventing a disease caused by Mycoplasma spp., comprising: an active ingredient, comprising a protein of EutD; and a pharmaceutically acceptable adjuvant; wherein said EutD comprises the sequence of SEQ ID NO: 10.

2. The composition of claim 1, wherein said active ingredient is of a concentration of 50 to 3500 g/mL based on the total volume of said composition.

3. The composition of claim 1, wherein said pharmaceutically acceptable adjuvant is a complete Freund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide, an oil emulsion, or a combination thereof.

4. The composition of claim 1, further comprising a pharmaceutically acceptable additive.

5. The composition of claim 4, wherein said pharmaceutically acceptable additive is a solvent, a stabilizer, a diluent, a preservative, an antibacterial agent, an antifungal agent, an isotonic agent, a absorption delaying agent, or a combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing will be provided by the USPTO upon request and payment of the necessary fee.

(2) FIG. 1 shows the result of the two-dimensional gel protein electrophoresis conducted in the 1.sup.st example of the present invention.

(3) FIG. 2 shows the result of the color reaction of the Western blot conducted in the 1.sup.st example of the present invention.

(4) FIG. 3 shows the result of the electrophoresis of the PCR products obtained in the 2.sup.nd example of the present invention.

(5) FIG. 4 shows the records of the challenge experiments conducted in the 3.sup.rd example of the present invention.

DESCRIPTION OF REFERENCE SIGNS IN THE FIGURES

(6) 1 XylF(xylose-binding lipoprotein) 2 XylF(xylose-binding lipoprotein) 3 XylF(xylose-binding lipoprotein) 4 PdhA(pyruvate dehydrogenase E1-alpha subunit) 5 Mhp145(periplasmic sugar-binding protein) 6 EutD(phosphotransacetylase) 7 EutD(phosphotransacetylase) 8 Mhp389 9 P78(lipoprotein) 10 P132

DETAILED DESCRIPTION OF THE INVENTION

(7) One of the core concepts of the present invention is to survey potential candidate antigens suitable for subunit vaccines by using two-dimensional gel protein electrophoresis along with immunological screening technology and to identify the antigens by mass spectrometer. Then, the performance of the present subunit vaccines were verified by animal model experiments.

(8) Briefly, the progress of the development of the present invention is:

(9) (1) Inducing immune response of experiment pigs by injecting a conventional M. hyopneumoniae vaccine and obtaining serum containing anti-M. hyopneumoniae antibodies. (2) Obtaining total proteins of M. hyopneumoniae for two-dimensional gel protein electrophoresis. (3) Conducting hybridization of the result of the two-dimensional gel protein electrophoresis of step (2) by using the serum of step (1) as 1.sup.st antibody, and then collecting proteins showing positive (i.e. candidate antigens) from the gel after amplification by a 2.sup.nd antibody and the following development procedure. (4) Identifying the candidate antigens obtained in step (3). (5) Expressing said candidate antigens in large amounts by using an E. coli gene expression system. (6) Examining the efficacy of the present subunit vaccines in reducing pathological traits in lung by swine challenge experiments and thereby verifying the value of said candidate antigens in being used as active ingredient of a subunit vaccine.

(10) The present vaccine for preventing Mycoplasma spp. infection comprises an active ingredient and a pharmaceutically acceptable adjuvant.

(11) In an embodiment of the present invention, said active ingredient may be PdhA, XylF, EutD, Mhp145, P78, P132, or Mhp389. In an alternative embodiment, as long as the antigenic determinant of any of the aforesaid protein is not interfered, said active ingredient may be a fusion protein of any two of the aforesaid proteins. In another alternative embodiment, said active ingredient comprises at least two of the aforesaid proteins; that is, so called a cocktail vaccine of the present invention.

(12) In another embodiment of the present invention, said active ingredient may comprise an amino acid sequence of SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or a combination thereof. In an alternative embodiment, as long as the antigenic determinant formed by folding of a peptide of said amino acid sequence is not interfered, said active ingredient may be a fusion protein with at least two said sequences. In another alternative embodiment, said active ingredient comprises two or more proteins respectively comprising one of the aforesaid amino acid sequences; that is, so called a cocktail vaccine of the present invention.

(13) Said pharmaceutically acceptable adjuvant is used for improving the immune effect of said active ingredient, stabilizing said active ingredient, and/or increasing the safety of vaccines. Said pharmaceutically acceptable adjuvant of the present invention includes, but not limits to: a complete Freund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide, an oil emulsion, or a combination thereof.

(14) The vaccine of the present invention may have one or at least two said active ingredients (i.e. a cocktail vaccine). In an example of the present vaccine, said active ingredient is of a concentration of 50 to 3500 g/mL based on the total volume of said vaccine. In a preferable embodiment of the present invention, when said vaccine comprises only one said active ingredient, said active ingredient is of a concentration of 50 to 500 g/mL based on the total volume of said vaccine. In an alternative embodiment of the present invention, the present vaccine comprises at least one said active ingredient; wherein the total concentration of said active ingredient(s) contained in said vaccine is 50 to 1000 g/mL, 50 to 1500 g/mL, 50 to 2000 g/mL, 50 to 2500 g/mL, 50 to 3000 g/mL, or 50 to 3500 g/mL based on the total volume of said vaccine.

(15) Another aspect of the present invention is to provide an expression vector for preventing Mycoplasma spp. infection. Specifically, said expression vector may be used for an E. coli gene expression system. Nevertheless, without being apart from the spirit of the present invention, those having ordinary skill in the art can modify said vector based on the disclosure of the present invention and make said vector suitable for different gene expression system while still belongs to the scope of the present invention.

(16) Said expression vector comprises a plasmid. Said plasmid comprises: a nucleotide sequence comprising at least one sequence selected from a group consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, and a combination thereof; and a regulatory element.

(17) Said vector is used in an E. coli gene expression system and for producing the antigens of the present invention via E. coli. In other words, said nucleotide sequence can be translated into the amino sequence of the present antigen via an E. coli gene expression system and then the amino acid sequence can fold into the present antigen.

(18) In an alternative embodiment, as long as the operation of the E. coli gene expression system is not hindered and the production of said nucleotide sequence and the folding of the consequent amino acid sequence thereof are not interfered, said plasmid may comprise two or more said nucleotide sequences.

(19) Said regulatory element is referred to an element required for initiating the transcription and translation in the expression system. Said regulatory element shall at least comprise a promoter, and a ribosome binding site. Preferably, said regulatory element may further comprise: an operator, an enhancer sequence, or a combination thereof.

(20) In a preferable embodiment of the present invention, said plasmid further comprises a gene encoding a fusion partner. Said fusion partner includes but not limits to msyB of E. coli, yjgD of E. coli, protein D of Lambda bacteriophage, or SUMO of S. cerevisiae. Said MsyB is rich in acidic amino acid and might be favorable for improving the solubility of the proteins to be produced.

(21) The following examples recite the trials and experiments of the present invention in order to further explain the features and advantages of the present invention. It shall be noted that the following examples are exemplary and shall not be used for limiting the claim scope of the present invention.

Example 1: Screening for Candidate Antigens Suitable for being Used as Active Ingredient of a Subunit Vaccine

(22) Preparation of Serum Containing Anti-Swine Mycoplasm Spp. Antibody.

(23) According to researches, there are seven Mycoplasm spp. can be isolated from swine: Mycoplasm hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma flocculare, Mycoplasma hyopharyngis, Mycoplasma sualvi, Mycoplasma bovigenitalium (Gourlay et al., 1978; Blank et al., 1996; Assuncao et al., 2005). Among them, M. hyopneumoniae is the major pathogen of swine enzootic pneumonia with an infection rate of 25 to 93%. Therefore, the present invention used M. hyopneumoniae (PRIT-5 strain) for immune proteomics studies and as sources of genes encoding antigens. Friis medium (Friis et al., 1975) as used for culturing M. hyopneumoniae. According to the experiment design, a proper amount of antibiotic or agar of 1.5% was added to formulating a solid medium.

(24) Three SPF pigs of 4-week old were brought from Agricultural Technology Research Institute and fed with same feed and kept at same environment and growth condition in piggery before experiments.

(25) After the pigs were fed to 32-day, 46-day, and 60-day old, the pigs were administrated 2 mL of Bayovac MH-PRIT-5 (M. hyopneumoniae PRIT-5) vaccine via intramuscular injection. Then, the pigs were continuously fed to 74-day old and blood was collected from a jugular vein thereof. The collected blood was placed in room temperature for 1 hour and stored in 4 C. In the next day, the collected blood was centrifugated at 1,107g for 30 minutes and the supernatant was removed to a clean tube and stored in 20 C.

(26) Two-Dimensional Gel Protein Electrophoresis of the Total Protein of Mycoplasm Spp.

(27) ReadyPrep protein extraction kit (total protein) (Bio-Rad, CA, USA) was used for extracting the total protein of Mycoplasm spp. Afterward, the concentration of the protein collected was determined by using a Bio-Rad RC DC Protein Assay Kit (CA, USA). The detailed protocol can be referred from the product description or can be modified from well-known protocols in the field.

(28) The two-dimensional gel protein electrophoresis was conducted in two steps: isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IEF was to separate proteins in the sample in view of isoelectric point thereof; SDS-PAGE was to separate proteins accordance with molecular weight thereof. Please see FIG. 1, which shows the result of the two-dimensional gel protein electrophoresis.

Hybridization

(29) The serum obtained in step (1) was used as 1.sup.st antibody to hybridize with the result of the two-dimensional gel protein electrophoresis in step (2). After being amplified by 2.sup.nd antibody and developed by the following development procedure, proteins showing positive were collected. Those proteins were recognized by the anti-Mycoplasm spp. antibody and therefore would be suitable as candidate antigens for active ingredient of subunit vaccines.

(30) The hybridization was conducted by Western blotting. Briefly, the 2D gel after electrophoresis was transferred to a PVDF membrane. Then, the membrane was incubated and hybridized sequentially with 1.sup.st antibody (the serum containing anti-Mycoplasm spp. antibody) and 2.sup.nd antibody (AP-conjugated anti-pig IgG). Afterward, a color reaction was conducted by using NBT/BCIP solution.

(31) The result of the color reaction of Western blotting was shown in FIG. 2; wherein 10 proteins positive to the immuno-hybridization with anti-Mycoplasm spp. antibody were marked as candidate antigens for being used as active ingredients of subunit vaccines.

Identification of the Candidate Antigens Obtained

(32) According to the color reaction of the Western blotting, the gel corresponding to the positive location on the membrane was cut by micropeptide and analyzed by mass spectrometry. The obtained data of the mass spectrometry was then matched with amino acid sequence and protein database to identify those proteins.

(33) Please see the following table 1, said 10 proteins positive to the immune-hybridization with anti-Mycoplasm spp. antibody were listed.

(34) TABLE-US-00001 TABLE 1 the 10 proteins positive to the immune-hybridization with anti-Mycoplasm spp. antibody and amino sequence thereof Candidate Name SEQ ID NO 1 XylF (xylose-binding lipoprotein) SEQ ID NO: 09 2 XylF (xylose-binding lipoprotein) SEQ ID NO: 09 3 XylF (xylose-binding lipoprotein) SEQ ID NO: 09 4 PdhA (pyruvate dehydrogenase SEQ ID NO: 08 E1-alpha subunit) 5 Mhp145 (periplasmic sugar-binding SEQ ID NO: 11 protein) 6 EutD (phosphotransacetylase) SEQ ID NO: 10 7 EutD (phosphotransacetylase) SEQ ID NO: 10 8 Mhp389 SEQ ID NO: 14 9 P78 (lipoprotein) SEQ ID NO: 12 10 P132 SEQ ID NO: 13 *XylF and EutD have different charge states in cells and therefore become 3 and 2 positive location on the membrane.

Example 2: Expressing of Said Candidate Antigens in Large Amount by E. coli Gene Expression System

(35) Escherichia coli JM109 was used as the host cells for cloning and Escherichia coli BL21 (DE3) was used as the host cells for protein expression. The Escherichia coli cells were cultured in LB medium (Luria-Bertani; Difco, Mich., USA). According to the experiment design, a proper amount of antibiotic or agar of 1.5% was added to formulating a solid medium.

Amplification of the Genes Encoding the Candidate Antigens

(36) After the candidate antigens were identified, the genes encoding those antigens were searched in the NCBI database (National Center for Biotechnology Information). Specific primers targeting the antigen genes were designed accordingly. Then, the antigen genes were amplified by using the specific primers and the chromosome of M. hyopneumoniae PRIT-5 as template. The specific primers used were listed in the following table 2.

(37) TABLE-US-00002 TABLE2 Primerset. Candidate Sequencesoftheprimerset PdhA PdhAF(SEQIDNO:15) 5-GATATAGGATCCATGGACAAATTTCG CTATGTAAAGCCTG-3 PdhAR(SEQIDNO:16) 5-CAATATGTCGACTTATTTTACTCCTT TAAAAAATTCAAGCGCTTC-3 XylF XylFF(SEQIDNO:17) 5-GATATAGGATCCATGAATGGAATAAA TTTCTTGGCTTAGGGTTAGTTTTTC-3 XylFR(SEQIDNO:18) 5-CAATATGTCGACTTAATTTTTATTAA TATCGGTAATTAGTTTGTCTAAGC-3 EutD EUTDF(SEQIDNO:19) 5-GATATAGGATCCATGACATACCAAGA ATATCTTCAAGCAAG-3) EUTDR(SEQIDNO:20) 5-CAATATGTCGACCTATTTACCTTCTT CAACTTGTAGAGCGCT-3) Mhp145 Mhp145F(SEQIDNO:21) 5-GATATAGGATGCATAGCTTGAAGGTC GAATACAACTGG-3 Mhp145R(SEQIDNO:22) 5-GAATATGTCGACTTAATTTACCTTTT GGAGTATGGGATTTTC-3 P78 P78F(SEQIDNO:23) 5-GATATAGGATCCTTATCCTATAAATT TAGGCGTTTTTTCC-3 P78R(SEQIDNO:24) 5-CAATATGTCGACTTATTTTGATTTAA AAGCAGGACCTAAAT-3 P132 P132F(SEQIDNO:25) 5-GATATAGGATCCATTGGACTAACAAT TTTTGAGAAATGATTTAG-3 P132R(SEQIDNO:26) 5-CAATATGTCGACTTATTCCTAAATAG CCCCATAAAGTG-3 Mhp389 Mhp389F(SEQIDNO:27) 5-GATATAGGATCCATGGACAAATTTTC ACGAACTGTTCT-3 Mhp389R(SEQIDNO:28) 5-CAATATGTCGACCTAGATTTTAAAGG ATTTTTTTAATTCAATAATATAATC-3

(38) Polymerase chain reaction (PCR) was conducted with the primer sets listed in the table 2 above to amplify the genes of the candidate antigens. The amplified genes were then used in the E. coli gene expression system. The PCR condition was: 5 minutes in 98 C. (one round); 30 seconds in 94 C., 30 seconds in 55 C., X seconds in 68 C. (35 rounds); 5 minutes in 68 C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. After the PCR reaction, an electrophoresis was conducted to verify if the PCR products contained the DNA fragments of expected size. Please see FIG. 3, which shows the electrophoresis result of the PCR products; wherein lane 1 was eutD gene; lane 2 was pdhA; lane 3 was xylF; lane 4 was P78 gene; lane 5 was P132 gene; lane 6 was mhp145; lane 7 was mhp389.

Cloning of the PCR Products

(39) The cloning was conducted by using a CloneJET PCR Cloning Kit, and the ligation mixture was transformed into E. coli ECOS 9-5 (Yeastern, Taipei, Taiwan). The detailed protocol can be referred from the product description or modified from the well-known protocol in the field.

(40) After transformation, the bacteria were cultured on a solid LB medium containing ampicillin (100 g/mL) until colony thereof formed. Then, colony PCR was conducted to screen strains success in transformation. The PCR condition was: 5 minutes in 95 C. (one round); 30 seconds in 95 C., 30 seconds in 55 C., X seconds in 72 C. (25 rounds); 7 minutes in 72 C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. The elongation speed of Taq DNA polymerase (Genomics, Taipei, Taiwan) is 1 kb/min; therefore, if Taq DNA polymerase is used for amplifying a 1 kb DNA fragment, said X shall be set as 1 minute.

(41) The plasmids of strains, whose recombinant plasmids were verified having the insert DNA, were then proceeded to DNA sequencing (Total Solution Provider of Systems Biology and Chemoinformatics Ltd.). Plasmids containing eutD, pdhA, xylF, P78 gene, P132 gene, mhp145, and mhp389 were named as pJET-eutD, pJET-pdhA, pJET-xylF, pJET-P78, pJET-P132, pJET-mhp145, pJET-mhp389, respectively.

(42) Point Mutation and Cloning of the Antigen Genes of M. Hyopneumoniae.

(43) Before amplifying the candidate antigens in an E. coli gene expression system, the codon usage in different organisms shall be considered. That said, if the gene contains codon that would be encoded ambiguously between the original organism therefrom and E. coli, the gene shall be modified by point mutation.

(44) The M. hyopneumoniae antigen genes, pdhA, xylF, P78 gene, P132 gene, mhp145, and mhp389, contain TGA codon (eutD does not have the concern in codon usage like others). The TGA codon was translated into tryptophan in Mycoplasma spp. but translated as stop codon in E. coli. In order to prevent from not being able to produce the entire protein in an E. coli gene expression system, primers targeting the TGA site were designed and point mutation replacing TGA with TGG was conducted by using overlapping extension polymerase chain reaction. As a result, the genes to be expressed in the E. coli gene expression system can be truthfully translated into the candidate antigen of the present invention. Besides, the cutting sites of BamHI of P78 gene, P132 gene, and mhp389 were undergone silent mutation for the convenience of cloning.

(45) The primers used for point mutation was designed to locate the site of point mutation at the central part of the primer and to have a Tm value of higher than 78 C. The Tm value of the primers for point mutation was calculated by using the formula provided by Invitrogene Co.: Tm=81.5+0.41 (% GC)675/N% mismatch; wherein % GC is referred as the percentage of GC in view of the total nucleotides contained in the primer concerned; N is referred as the length of the primer concerned; % mismatch is referred as the percentage of the base to be mutated in view of the total nucleotides contained in the primer concerned. The primer sets used for the aforesaid genes were listed in the following Table 3 to Table 8.

(46) TABLE-US-00003 TABLE3 TheprimersetsforpointmutationofpdhA. Primer DNAsequence(5 to3) PdhAF GATATAGGATCCATGGACAAATTTCGCTATGTAAAG SEQIDNO:29 CCTG PdhAM1 GCTAACAAAAGATGACTGGTTTGTCCCAGCTTTTCG SEQIDNO:30 PdhAM2 CGAAAAGCTGGGACAAACCAGTCATCTTTTGTTAGC SEQIDNO:31 PdhAM3 CTTGCAAATGCAATATTGGAATGGTAGCGAAAAAGG SEQIDNO:32 PdhAM4 CCTTTTTCGCTACCATTCCAATATTGCATTTGCAAG SEQIDNO:33 PdhAM5 CGAGGCGCTAAATATTGCAAGTATTTGGAAATGGCC SEQIDNO:34 AGTTGTTTTTTGCGTAAATAAC PdhAM6 GTTATTTACGCAAAAAACAACTGGCCATTTCCAAAT SEQIDNO:35 ACTTGCAATATTTAGCGCCTCG PdhAM7 GTTTTTTGCGTAAATAACAATCAATGGGCAATTTCA SEQIDNO:36 ACCCCAAATAAATATG PdhAM8 CATATTTATTTGGGGTTGAAATTGCCCATTGATTGT SEQIDNO:37 TATTTACGCAAAAAAC PdhAM9 GTTGAGTTTGTAACTTGGCGTCAAGGTGTTCATACC SEQIDNO:38 PdhAM10 GGTATGAACACCTTGACGCCAAGTTACAAACTCAAC SEQIDNO:39 PdhAM11 GAGAACACGAAAAATGGGAACCAATGCACCGG SEQIDNO:40 PdhAM12 CCGGTGCATTGGTTCCCATTTTTCGTGTTCTC SEQIDNO:41 PdhAM13 CCGAAAAACAAAAAATTTGGGATGAAGCGCTTGCGA SEQIDNO:42 TTG PdhAM14 CAATCGCAAGCGCTTCATCCCAAATTTTTTGTTTTT SEQIDNO:43 CGG PdhAR CAATATGTCGACTTATTTTACTCCTTTAAAAAATTC SEQIDNO:44 AAGCGCTTC

(47) TABLE-US-00004 TABLE4 TheprimersetsforpointmutationofxylF. Primer DNAsequence(5 to3) XylFF GATATAGGATCCATGAAATGGAATAAATT SEQIDNO:45 TCTTGGCTTAGGCTTAGTTTTTC XylFM1 CATTTAACCAATCAAGTTGGGAGGCAATT SEQIDNO:46 CAACAACTTGG XylFM2 CCAAGTTGTTGAATTGCCTCCCAACTTGA SEQIDNO:47 TTGGTTAAATG XylFM3 CTAATACCAACAAAAATGTTTGGGTACTT SEQIDNO:48 TCTGGTTTTCAACACG XvlFM4 CGTGTTGAAAACCAGAAAGTACCCAAACA SEQIDNO:49 TTTTTGTTGGTATTAG XylFM5 CGGTGATGCGATCACAAAATGGTTAAAAA SEQIDNO:50 TCCCTGAAAATAAGC XylFM6 GCTTATTTTCAGGGATTTTTAACCATTTT SEQIDNO:51 GTGATCGCATCACCG XylFM7 TTATCATACTCGGAATTGACTGGACTGAT SEQIDNO:52 ACTGAAAATGTAATTC XylFM8 GAATTACATTTTCAGTATCAGTCCAGTCA SEQIDNO:53 ATTCCGAGTATGATAA XylFM9 GAAGAAGCCGGATGGCTTGCAGGATATGC SEQIDNO:54 XylFM10 GCATATCCTGCAAGCCATCCGGCTTCTTC SEQIDNO:55 XylFM11 GGTTATCTAGCCGGAATTAAAGCTTGGAA SEQIDNO:56 TCTAAAAAATTCTGATAAAAAAAC XylFM12 GTTTTTTTATCAGAATTTTTTAGATTCCA SEQIDNO:57 AGCTTTAATTCCGGCTAGATAACC XylFR CAATATGTCGACTTAATTTTTATTAATAT SEQIDNO:58 CGGTAATTAGTTTGTCTAAGC

(48) TABLE-US-00005 TABLE5 TheprimersetsforpointmutationofP78gene. Primer DNAsequence(5 to3) P78F GATATAGGATCCTTATCCTATAAATTTAGG SEQIDNO:59 CGTTTTTTCC P78M1 CAATTAATAAAGTTTTGTTTGGTTGGATGA SEQIDNO:60 TTAATAAAGCACTTGCTGATCC P78M2 GGATCAGCAAGTGCTTTATTAATCATCCAA SEQIDNO:61 CCAAACAAAACTTTATTAATTG P78M3 GATATTAAAGAAATTGAAAGAATCTGGAAA SEQIDNO:62 AAATATGTCTCCGATGATCAAGG P78M4 CCTTGATCATCGGAGACATATTTTTTCCAG SEQIDNO:63 ATTCTTTCAATTTCTTTAATATC P78M5 GCCCTTTCAGGAGGCTCCACTGATTCGGCA SEQIDNO:64 P78M6 TGCCGAATCAGTGGAGCCTCCTGAAAGGGC SEQIDNO:65 P78M7 GCCGCAAAAGCTTTTGTTAAATGGCTTTTG SEQIDNO:66 ACAGAAAAAATAGTCT P78M8 AGACTATTTTTTCTGTCAAAAGCCATTTAA SEQIDNO:67 CAAAAGCTTTTGCGGC P78R CAATATGTCGACTTATTTTGATTTAAAAGC SEQIDNO:68 AGGACCTAAAT

(49) TABLE-US-00006 TABLE6 TheprimersetsforpointmutationofP132gene. Primer DNAsequence(5 to3) P132F GATATAGGATCCATTGGACTAACAATTTTT SEQIDNO:69 GAGAAATCATTTAG P132M1 CTAACTTCTCTAAAAGGTTGGAAAGAAGAA SEQIDNO:70 GATGATTTTG P132M2 CAAAATCATCTTCTTCTTTCCAACCTTTTA SEQIDNO:71 GAGAAGTTAG P132M3 CTTTCTATTACTTTTGATCTCTGGGACCCA SEQIDNO:72 AATGGTAAATTAGTATC P132M4 GATACTAATTTACCATTTGGGTCCCAGAGT SEQIDNO:73 TCAAAAGTAATAGAAAG P132M5 CCCTGAAGGAGATTGGATAACTTTAGGGAG SEQIDNO:74 P132M6 CTCCCTAAAGTTATCCAATCTCCTTCAGGG SEQIDNO:75 P132M7 CTACCAGGAACTACCTGGGATTTCCATGTT SEQIDNO:76 GAAC P132M8 GTTCAACATGGAAATCCCAGGTAGTTCCTG SEQIDNO:77 GTAG P132M9 GGACAACTAATTTGGAGCCAGTTAGCTTCC SEQIDNO:78 P132M10 GGAAGCTAACTGGCTCCAAATTAGTTGTCC SEQIDNO:79 P132M11 GGAACAAAAAAGGAATGGATTCTTGTAGGA SEQIDNO:80 TCTGG P132M12 CCAGATCCTACAAGAATCCATTCCTTTTTT SEQIDNO:81 GTTCC P132M13 CCAATACGCAAATATGGATAACCCGTCTAG SEQIDNO:82 GAAC P132M14 GTTCCTAGACGGGTTATCCATATTTGCGTA SEQIDNO:83 TTGG P132M15 CCAAGGGGAAGTTCTCTGGACTACTATTAA SEQIDNO:84 ATCCAAAC P132M16 GTTTGGATTTAATAGTAGTCCAGAGAACTT SEQIDNO:85 CCCCTTGG P132M17 CAAAAAACTTCACCTTTGGTGGATTGCTAA SEQIDNO:86 TGATAGC P132M18 GCTATCATTAGCAATCCACCAAAGGTGAAG SEQIDNO:87 TTTTTTG P132R CAATATGTCGACTTATTCCTAAATAGCCCC SEQIDNO:88 ATAAAGTG

(50) TABLE-US-00007 TABLE7 Theprimersetsforpointmutationofmhp145. Primer DNAsequence(5 to3) Mhp145F GATATAGGATCCATAGCTTCAAGGTCGAATACAA SEQIDNO:89 CTGC Mhp145M1 AATAATTGCAGAAAAAATTCTTAAAGATCAATGG SEQIDNO:90 AAAACAAGTAAATATTCTGATTTTTATTCACAAT Mhp145M2 ATTGTGAATAAAAATCAGAATATTTACTTGTTTT SEQIDNO:91 CCATTGATCTTTAAGAATTTTTTCTGCAATTATT Mhp145R CAATATGTCGACTTAATTTACCTTTTGGAGTATC SEQIDNO:92 CCATTTTC

(51) TABLE-US-00008 TABLE8 Theprimersetsforpointmutationofmhp389. Primer DNAsequence(5 to3) Mhp389F GATATAGGATCCATGGACAAATTTTCAC SEQIDNO:93 GAACTGTTCT Mhp389M1 CAATAGTGACAATGGACCCCCCAAATGT SEQIDNO:94 TGGTCG Mhp389M2 CGACCAACATTTGGGGGGTCCATTGTCA SEQIDNO:95 CTATTG Mhp389M3 GATAAAGGCGCATCATGGGTTGCGCTTG SEQIDNO:96 CACGAAC Mhp389M4 GTTGGTGCAAGCGCAAGCCATGATGCGC SEQIDNO:97 CTTTATC Mhp389M5 GGAAAACTTAAAGGTAAATGGACTTTTG SEQIDNO:98 GACTAACCTATTT Mhp389M6 AAATAGGTTAGTCCAAAAGTCCATTTAC SEQIDNO:99 CTTTAAGTTTTCC Mhp389R CAATATGTCGACCTAGATTTTAAAGGAT SEQIDNO:100 TTTTTTTAATTCAATAATATAATC

(52) The method for the point mutation was briefly explained as follows. The chromosome of M. hyopneumoniae PRIT-5 was used as template and DNA fragments was amplified by using the primer sets set forth in the table 3 to table 8 above.

(53) The 50 L PCR reaction mixture comprised 1GDP-HiFi PCR buffer, 200 M of mixture of dATP, dTTP, dGTP, and dCTP, 1 M of primers, 100 ng of chromosome of M. hyopneumoniae PRIT-5, and 1 U of GDP-HiFi DNA polymerase. The PCR condition was: 5 minutes in 98 C. (one round); 30 seconds in 94 C., 30 seconds in 55 C., X seconds in 68 C. (35 rounds); 5 minutes in 68 C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. The elongation speed of GDP-HIFI DNA polymerase (GeneDirex, Las Vegas, USA) is 1 kb/15 seconds; therefore, if GDP-HIFI DNA polymerase is used for amplifying a 1 kb DNA fragment, said X shall be set as 15 seconds. After the PCR reaction, an electrophoresis was conducted to verify if the PCR products contained the DNA fragments of expected size. Then, the PCR product was recycled by using a Gel-M gel extraction system kit.

(54) Afterward, the PCR product was used as template and amplified by using the primer sets set forth in the table 2 above. The PCR condition was: 2 minutes in 98 C. (one round); 30 seconds in 94 C., 30 seconds in 55 C., X seconds in 68 C. (35 rounds); 5 minutes in 68 C. (one round). Said X was the elongation time for the DNA polymerase and was set depending on the size of the fragment to be amplified. The elongation speed of GDP-HIFI DNA polymerase (GeneDirex, Las Vegas, USA) is 1 kb/15 seconds; therefore, if GDP-HIFI DNA polymerase is used for amplifying a 1 kb DNA fragment, said X shall be set as 15 seconds. After the aforesaid amplification step, a full length sequence of the candidate antigen genes with point mutation can be obtained.

(55) Then, the PCR product was recycled by using a PCR-M Clean Up system kit (GeneMark, Taichung, Taiwan) and the cloning thereof was conducted by using a CloneJET PCR Cloning Kit. Colony PCR was conducted to confirm the strains after transformation containing plasmid having the insert DNA and then the plasmids therein were isolated for DNA sequencing (Total Solution Provider of Systems Biology and Chemoinformatics Ltd.). Plasmids containing mutated candidate antigen genes were named as pJET-pdhAM, pJET-xylFM, pJET-P78M, pJET-P132M, pJET-mhp145M, pJET-mhp389M, respectively.

(56) According to the result of sequencing, the DNA sequences of the candidate antigen genes after point mutation were as shown in SEQ ID NO:01 (pdhA), SEQ ID NO:02 (xylF), SEQ ID NO:03 (eutD, was not point-mutated), SEQ ID NO:04 (mhp145), SEQ ID NO:05 (P78 gene), SEQ ID NO:06 (P132 gene), SEQ ID NO:07 (mhp389).

(57) Construction of the Expression Vectors for Expressing the M. Hyopneumoniae Antigens

(58) In this part of experiments, plasmid pET-MSY was used as backbone for constructing an expression vector for expressing M. hyopneumoniae antigen. pET-MSY is a derivative of pET29a and has a E. coli msyB. Therefore, the expressed recombinant antigen thereby would have a fusion partner MsyB. MsyB is rich in acidic amino acid and is able of increasing the solubility of the protein expressed.

(59) After pJET-eutD, pJET-pdhA, pJET-xylF, pJET-P78, pJET-P132, pJET-mhp145 and pJET-mhp389 being digested by BamHI and SalI, DNA fragment obtained was inserted into pET-Msy digested previously with the same restriction enzymes by ligase. Then, the pET-Msy with the DNA fragment was transformed into E. coli ECOS 9-5. Colony PCR was conducted to confirm the strains after transformation containing plasmid having the insert DNA and then the plasmids therein were isolated for DNA sequencing (Total Solution Provider of Systems Biology and Chemoinformatics Ltd.). Plasmids verified with correct DNA sequence were named as pET-MSYEutD, pET-MSYPdhA, pET-MSYXy1F, pET-MSYP78, pET-MSYP132, pET-MSYMhp145, and pET-MSYMhp389, respectively. Those plasmids obtained were examples of the expression vectors for preventing Mycoplasma spp. infection of the present invention.

Expression and Isolation of the M. hyopneumoniae Antigens

(60) The vectors for antigen expression were transformed into E. coli BL21 (DE3). Single colony of consequent strains after transformation was inoculated in LB liquid medium containing kanamycin (working concentration: 30 g/mL). After culture overnight at 37 C., 180 rpm, the suspension of the bacteria was diluted at ratio of 1:100 and inoculated again in another LB liquid medium containing kanamycin (working concentration: 30 g/mL). The bacteria were cultured at 37 C., 180 rpm until OD.sub.600 therefore achieving about 0.6 to 0.8. Then, 0.1 mM of IPTG was added to induce expression. After induction for 4 hours, pellet was collected by centrifugation (10000g, 10 minutes, 4 C.) and the expression was examined via protein electrophoresis.

(61) Afterward, immobilized-metal affinity chromatography (IMAC) was used for protein isolation through the covalent bonding between the His tag of the N-terminal of the recombinant protein and nickel ions or cobalt ions. The protocol of protein isolation was in accordance with the product description of the QIAexpressionist (fourth edition, Qiagen). The pellet was suspended in a lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and disturbed by an ultrasonic processer. After centrifugation (8,000g, 15 minutes), the supernatant was collected to introduce into a column of 1 mL Ni-NTA resin. The recombinant antigens would adhere on said resin. Then, 15 mL wash buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole, pH 8.0) was introduced into the column to wash the resin so that nonspecific proteins adhering thereon can be removed. Lastly, 20 mL elution buffer was added (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM imidazole, pH 8.0) to wash off the recombinant antigens on the resin; wherein the imidazole of high concentration can compete the binding site on the resin with the recombinant proteins and thereby cause the recombinant proteins being washed off. The result of isolation was then examined by protein electrophoresis.

(62) The candidate antigens of the present invention collected by isolation can then be used for the following immune trials to confirm their ability to be used as active ingredient of anti-Mycoplasm spp. subunit vaccines.

Example 3: Swine Immune Challenge Experiments of the Candidate Antigens of the Present Invention

(63) In this example, the candidate antigens of the present invention were used as active ingredient for preparing subunit vaccines and tested for immune effects thereof in live swine.

Vaccine Preparation

(64) One isolated recombinant antigen or several isolated recombinant antigens were mixed with alumina gel as an adjuvant to prepare a subunit vaccine or a cocktail subunit vaccine. Every dose of the prepared vaccine was of 2 mL in volume and each kind of antigen contained therein was of 100 g.

(65) The following table 9 listed the samples prepared in this example for immune challenge experiments.

(66) TABLE-US-00009 TABLE 9 Samples of vaccine prepared in Example 3 Sample Active Ingredient (Antigen) 1 PdhA 2 XylF 3 EutD 4 Mhp145 5 P78 6 P132 7 Mhp389 8 PdhA + P78 9 XylF + Mhp145

(67) The swine immune challenge experiments would be conducted by using Bayovac MH-PRIT-5 (made by using M. hyopneumoniae PRIT-5, as a positive control group), subunit vaccines (samples 1-7 of the present invention), and cocktail vaccines (samples 8 and 9 of the present invention).

(68) 33 SPF pigs of 4-week old were brought from Agricultural Technology Research Institute and fed with same feed, environment, and growth condition in piggery before experiments.

(69) After the pigs were fed to 35-day and 49-day old, the pigs were administrated 2 mL of vaccine above via intramuscular injection.

Challenge Experiments

(70) The aforesaid pigs being induced immune response were challenged by Mycoplasm spp. at 109-day old to confirm the immune effect of the aforesaid vaccines.

(71) First of all, a lung collected from pigs infected by Mycoplasm spp. was ground in 20 mL of Friis medium and centrifugated at 148.8g for 10 minutes. The supernatant was removed to a clean tube and centrifugated again at 7,870g for 40 minutes. Then, the supernatant was discarded and the precipitation was suspended in 6 mL of Friis medium to obtain a suspension. Afterward, the suspension was filtered by membrane of 5 m and 0.45 M sequentially to obtain bacteria solutions required for the challenge experiments.

(72) The bacteria solution (5 mL) was administrated to narcotized pigs via trachea thereof. After 28 days from administration, the pigs were sacrificed and dissected to collect lung thereof. The immune effect was examined by observing the lung and recorded according to the following criteria: any of meddle upper lobes and upper lobes of any side of the lung observed of pathological trait was scored as 10 points; any of meddle upper lobe and diaphragmatic lobes of any side of the lung observed of pathological trait was scored as 5 points. The full score was 55 points. The observation records were shown in FIG. 4.

(73) In comparison with the results of non-injected pigs, the seven candidate antigens of the present invention were able to provide equivalent immune effects as conventional vaccine (Bayovac MH-PRIT-5). If the higher safety of subunit vaccines is taking into consideration, the vaccines containing the candidate antigens of the present invention shall be valued more.

(74) On the other hand, it was not common to use two or more antigens that would induce immune effects in one vaccine because the two or more antigens may not provide doubled immune effect. In fact, there is higher chance that the two or more antigens may interfere or against each other and consequently reduce the immune effect of the vaccine. According to the result of this example, sample 8 and sample 9 of the present invention (i.e. cocktail vaccine) unexpectedly provide significant increase in the immune effect. That said, the subunit vaccines of the present invention not only have high safety but also provide better immune effect when the candidate antigens of the present invention are used in combination.

(75) Those having ordinary skill in the art can readily understand any possible modifications based on the disclosure of the present invention without apart from the spirit of the present invention. Therefore, the examples above shall not be used for limiting the present invention but intend to cover any possible modifications under the spirit and scope of the present invention according to the claims recited hereinafter.