CONSTRUCTION AND APPLICATION OF ONE INNOVATIVE EXPRESSION VECTOR FOR VIRUS-LIKE PARTICLES

Abstract

The present invention provides a novel virus-like particle expression vector pTMSCA2C, which is constructed as follows: firstly, using plasmid pTrcHis-MS2 as a starting vector and mutating the base T at position 5 of the gene sequence of MS2 bacteriophage 19mer packaging site on the plasmid pTrcHis-MS2 into C through genetic mutation technologies to obtain a plasmid pTMSC; then, mutating valine which is a amino acid corresponding to the initiation codon on the plasmid pTMSC for encoding the maturase protein of MS2 bacteriophage into methionine to obtain a plasmid pTMSCA; and finally, the gene sequence coding wild type MS2 bacteriophage coat protein, after the removal of the terminator, is linked in series with the gene sequence coding MS2 bacteriophage coat protein comprising histidine-tag which is from a pseudovirus vector pTrcMS, and the gene sequence obtained after linking in series is linked to the plasmid pTMSCA to give pTMSCA2C. When the virus-like particle is prepared by using the expression vector pTMSCA2C of the present invention, the yield and purity of the virus-like particle may be improved while the workload for preparation of virus-like particles may be greatly reduced.

Claims

1. A novel virus-like particle expression vector pTMSCA2C, characterized in that the expression vector pTMSCA2C is constructed as follows: using plasmid pTrcHis-MS2 as a starting vector and mutating the base T at position 5 of the gene sequence of MS2 bacteriophage 19mer packaging site on the plasmid pTrcHis-MS2 into C through genetic mutation technologies to obtain a plasmid pTMSC; then mutating valine which is an amino acid corresponding to the initiation codon on the plasmid pTMSC for encoding the maturase protein of MS2 bacteriophage into methionine to obtain a plasmid pTMSCA; and finally, the gene sequence coding wild type MS2 bacteriophage coat protein, after the removal of the terminator, is linked in series with the gene sequence coding MS2 bacteriophage coat protein comprising histidine-tag which is from a pseudovirus vector pTrcMS, and the gene sequence obtained after linking in series is linked to the plasmid pTMSCA to give pTMSCA2C; wherein, the nucleotide sequence of the plasmid pTrcHis-MS2 is set forth in SEQ ID NO: 1, the nucleotide sequence of the pseudovirus vector pTrcMS is set forth in SEQ ID NO: 2 and the nucleotide sequence of the expression vector pTMSCA2C is set forth in SEQ ID NO: 3.

2. (canceled)

3. A method for constructing the expression vector of claim 1, characterized in that the method comprises the following steps: 1) preparation of the plasmid pTMSC: using the plasmid pTrcHis-MS2 as a template, PCR amplification is performed with Primer A and Primer B into which a base mutation is introduced respectively to obtain PCR product A and PCR product B, and then the amplified products are recovered and purified; the plasmid pTrcHis-MS2 is subjected to double restriction enzyme digestion with XhoI and HindIII and the digested plasmid pTrcHis-MS2 is linked with the PCR product A and PCR product B by In-Fusion technique and transformed into a recipient; and after screening and identification, the plasmid pTMSC is obtained; 2) preparation of the plasmid pTMSCA: using the plasmid pTMSC as a template, PCR amplification is performed with Primer 1 into which a base mutation is introduced to obtain PCR product I, and then the amplified product is recovered and purified; the plasmid pTMSC is subjected to double restriction enzyme digestion with NcoI and PmaCI and the digested plasmid pTMSC is linked with the PCR product I by In-Fusion technique and transformed into a recipient; and after screening and identification, the plasmid pTMSCA is obtained; and 3) construction of the expression vector pTMSCA2C: a gene sequence coding wild type MS2 bacteriophage coat protein, after the removal of terminator, is linked in series with a gene sequence coding MS2 bacteriophage coat protein comprising histidine-tag from the pseudovirus vector pTrcMS, and the gene sequence obtained after linking in series is inserted into the plasmid pTMSCA between XhoI and HindIII restriction enzyme cutting sites to give the expression vector pTMSCA2C; wherein, the sequences of the Primer A, Primer B and Primer 1 are as follows: TABLE-US-00005 PrimerA-F: (SEQIDNO8) 5-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3 PrimerA-R: (SEQIDNO5) 5-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3; PrimerB-F: (SEQIDNO6) 5-GCCATTCAAACATGAGGATCACCCATGTCGAAG-3; PrimerB-R: (SEQIDNO7) 5-GTTCGGGCCCAAGCTTCGAATTCCC-3; Primer1-F: (SEQIDNO8) 5-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3; and Primer1-R: (SEQIDNO9) 5-CCACCTGCCGGCCACGTGTTTTGATC-3.

4. The method of claim 3, characterized in that, the primers used for identification in steps 1) and 2) are: TABLE-US-00006 Primer-U1: (SEQIDNO10) 5-GACAATTAATCATCCGGCTCG-3, and Primer-L1: (SEQIDNO11) 5-GATCTTCGTTTAGGGCAAGGTAG-3.

5. A virus-like particle, characterized in that the virus-like particle comprises a RNA transcript of an exogenous gene carried by the novel virus-like particle expression vector pTMSCA2C of claim 1.

6. A method for the preparation of virus-like particles, characterized in that, the method comprises the following steps: an exogenous gene fragment is cloned into the downstream of the gene sequences coding MS2 bacteriophage coat protein linked in series in the novel virus-like particle expression vector pTMSCA2C of claim 1; then, a terminator is inserted into the downstream of the exogenous gene fragment; after transcription, RNA transcripts of the exogenous gene carrying a RNA sequence of a bacteriophage operator are obtained; bacteriophage coat proteins are expressed after induction and assembled into protein coats while the RNA transcripts of the carried exogenous gene are encapsulated into the protein coats to give the virus-like particles.

7. (canceled)

8. A quality control product prepared from the virus-like particle of claim 5, used for the detection of pathogenic microorganisms.

9. A method for the preparation of a quality control product for the detection of pathogenic microorganisms which comprises using the virus-like particle expression vector of claim 1.

10. A method for the preparation of a quality control product for the detection of pathogenic microorganisms which comprises using the virus-like particle expression vector of claim 5.

11. A quality control product prepared from the virus-like particle of claim 1, used for the detection of pathogenic microorganisms.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 shows the comparison of nucleic acid electrophoresis on lysate supernatants of the expressed products (after induction) of the recombinant strain containing the plasmid pTMSCA and the recombinant strain containing the plasmid pTrcHis-MS2 in Example 1 of the present invention. Wherein, lane 1 was the lysate supernatant (5 L) of the expressed products of the recombinant strain containing the plasmid pTMSCA after induction; lane 2 was the lysate supernatant (5 L) of the expressed products of the recombinant strain containing the plasmid pTrcHis-MS2 after induction; and M was DL2000 DNA Marker.

[0026] FIG. 2 shows the comparison of nucleic acid electrophoresis on lysate supernatants of the expressed products after packaging exogenous fragments of different length by the recombinant strain containing the plasmid pTMSCA and the recombinant strain containing the plasmid pTrcHis-MS2 in Example 1 of the present invention. Wherein, M is DL2000 DNA Marker; lane 1 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment A (A<500 bp) by the recombinant strain containing the plasmid pTrcHis-MS2; lane 2 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment B (1,000 bp>B >500 bp) by the recombinant strain containing the plasmid pTrcHis-MS2; lane 3 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment C (1,500 bp>C>1,000 bp) by the recombinant strain containing the plasmid pTMSCA; and lane 4 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment D (D>1,800 bp) by the recombinant strain containing the plasmid pTMSCA.

[0027] FIG. 3 shows the comparison of nucleic acid electrophoresis on lysate supernatants of the expressed products after packaging exogenous fragments of different length by the novel virus-like particle expression vector pTMSCA2C, the recombinant strain containing the plasmid pTMSCA and the vector pTrcMS in Example 2 of the present invention. Wherein, M was 1 kb DNA Marker; lane 1 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment D (D>1,800 bp) by pTMSCA; lane 2 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment A (A<500 bp) by pTrcMS; lane 3 was the lysate supernatant (5 L) of the expressed products after packaging a exogenous gene fragment D (D>1,800 bp) by pTMSCA2C.

[0028] FIG. 4 shows the results of SDS-PAGE electrophoresis of the purified virus-like particle pTMSCA2C-SBV in Example 3. Wherein, lane 1 was the pTMSCA2C-SBV virus-like particle (Coomassie blue staining), and M was Spectra Multicolor Low Range Protein Ladder.

[0029] FIG. 5 is a fluorescence image of real time PCR detection of the purified virus-like particle pTMSCA2C-SBV in Example 3 of the present invention for the purpose of determining whether or not DNA residue is present.

[0030] FIG. 6 is an electron microscopy image of the virus-like particle pTMSCA2C-SBV in Example 3 of the present invention (100,000JEM1400).

BEST MODE

[0031] Without limitation on the scope of the present invention, the following Examples are for the purpose of explaining the present invention. Unless specified otherwise, the Examples are performed under conventional experimental conditions such as those recited in Molecular Cloning: A Laboratory Manual (Sambrook J & Russell D W, 2001) or under conditions suggested in the manufacturer's instructions.

EXAMPLE 1

Construction of a Recombinant Strain Containing a Plasmid pTMSCA

[0032] Firstly, using the plasmid pTrcHis-MS2 (SEQ ID NO: 1) as a template, the base T at position 5 of the gene sequence of MS2 bacteriophage 19mer packaging site was mutated into C by In-Fusion technology to give pTMSC; then, valine which was a amino acid corresponding to the initiation codon for encoding the maturase protein of MS2 bacteriophage was mutated into methionine to give pTMSCA.

[0033] The specific construction method is described as follows:

[0034] {circle around (1)} The base mutation was introduced into the PCR primers; using the plasmid pTrcHis-MS2 as a template, PCR amplification was performed by Primer STAR HS DNA Polymerase with the Primer A primer pair and the Primer B primer pair respectively to give amplification product named PCR product A and PCR product B, wherein, the PCR amplification primers were shown in SEQ ID NOs: 4-7:

TABLE-US-00003 PrimerA-F 5-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3; PrimerA-R 5-TGGGTGATCCTCATGTTTGAATGGCCGGCGTC-3; PrimerB-F 5-GCCATTCAAACATGAGGATCACCCATGTCGAAG-3; and PrimerB-R 5-GTTCGGGCCCAAGCTTCGAATTCCC-3.

[0035] 50 L PCR amplification reaction system is as follows: 5Prime STAR Buffer (Mg.sup.2+ plus), 10 L; Prime STAR HS DNA polymerase, 1.3 U; dNTP Mixture (each 2.5 mM), 4 L; Primer A-F/R (20 pmol/L), each 0.5 L; Primer B-F/R (20 pmol/L), each 0.5 L; pTrcHis-MS2 DNA template, 10 ng; add ddH.sub.2O to 50 L. PCR amplification program: 30 cycles of denaturation at 98 C. for 10 s, annealing at 55 C. for 10 s, extension at 72 C. for 30 s; and extension at 72 C. for 10 min. The amplification product was recovered and purified.

[0036] {circle around (2)} The plasmid pTrcHis-MS2 was subjected to double restriction enzyme digestion with XhoI and HindIII. The digestion system is as follows: XhoIIHindIII, 10 U; 10restriction enzyme digestion buffer, 5 L; plasmid template, 1 g; add ddH.sub.2O to 10 L. The reaction was performed at 37 C. for 2 hours. The product of the restriction enzyme digestion was subjected to gel recovery and purification respectively, and named as pTrcHis-MS2 (X/H).

[0037] {circle around (3)} Using In-Fusion HD Cloning Kit (Clontech Code No. 639648), PCR product A, PCR product B, and pTrcHis-MS (X/H) were linked together. The reaction system and conditions are as follows: PCR product A/B, each 200 ng; pTrcHis-MS2 (X/H), 100 ng; 5In-Fusion HD Enzyme Premix 2 L, add ddH.sub.2O to 10 L; the reaction was performed at 50 C. for 15 minutes.

[0038] {circle around (4)} 2.5 L of the above-mentioned In-Fusion product was taken and transformed into E. coli Competent Cell JM109 through heat-shock transformation. The obtained product was cultured overnight at 37 C. The positive clones were picked and identified by sequencing. The primers used for identification were:

[0039] Primer-U1: 5-GACAATTAATCATCCGGCTCG-3, and Primer-L1: 5-GATCTTCGTTTAGGGCAAGGTAG-3 (SEQ ID NOs: 10-11). The plasmid identified as having the correct sequence was named pTMSC.

[0040] {circle around (5)} The mutated base was introduced into the PCR primers; using the plasmid pTMSC as a template, PCR amplification was performed by Primer STAR HS DNA Polymerase with the Primer 1 as a primer pair to give amplification product named PCR product I, wherein, the PCR amplification primers were shown in SEQ ID NOs: 8-9:

TABLE-US-00004 Primer1-F: 5-GAGGAATAAACCATGCGAGCTTTTAGTACCCTTG-3, and Primer1-R: 5-CCACCTGCCGGCCACGTGTTTTGATC-3.

[0041] 50 L PCR amplification reaction system is as follows: 5Prime STAR Buffer (Mg.sup.2+ plus), 10 L; Prime STAR HS DNA polymerase, 1.3 U; dNTP Mixture (each 2.5 mM), 4 L; PrimerI-F/R (10 pmol/L), each 0.5 L; pTMSC DNA template, 10 ng; add ddH.sub.2O to 50 L. PCR amplification program: 30 cycles of denaturation at 98 C. for 10 s, annealing at 55 C. for 10 s, extension at 72 C. for 30 s; and extension at 72 C. for 10 min. The amplification product was recovered and purified.

[0042] {circle around (6)} The plasmid pTMSC was subjected to double restriction enzyme digestion with NcoI and PmaCI. The digestion system is as follows: NcoIIPmaCI, 10 U; 10restriction enzyme digestion buffer, 5 L; plasmid template, 1 g; add ddH.sub.2O to 10 L. The reaction was performed at 37 C. for 2 hours. The product of the restriction enzyme digestion was subject to gel recovery and purification respectively, and named as pTMSC (N/P).

[0043] {circle around (7)} Using In-Fusion HD Cloning Kit (Clontech Code No. 639648), the PCR product I and pTMSC (N/P) were linked together. The reaction system and conditions are as follows: PCR product I, 200 ng; pTMSC (N/P), 100 ng; 5In-Fusion HD Enzyme Premix 2 L, add ddH.sub.2O to 10 L; the reaction was performed at 50 C. for 15 minutes.

[0044] {circle around (8)} 2.5L of the above-mentioned In-Fusion product was taken and transformed into E. coli Competent Cell JM109 through heat-shock transformation. The obtained product was cultured overnight at 37 C. The positive clones were picked and identified by sequencing. The primers used for identification were as follows:

[0045] Primer-U1: 5-GACAATTAATCATCCGGCTCG-3, and Primer-L1: 5-GATCTTCGTTTAGGGCAAGGTAG-3. The plasmid identified as having the correct sequence was named as pTMSCA.

[0046] {circle around (9)} Comparison results between the lysate supernatants of pTMSCA and pTrcHis-MS2: the recombinant strains pTMSCA and pTrcHis-MS2 were respectively induced by IPTG (final concentration, 1 mol/L) to express for 16 hours, centrifugated at 5,000 rpm for 10 minutes to collect cells. The product precipitate was added with 20 L 1TE buffer (pH 8.0) per mL cell precipitate, mixed well by vortex, added with 1 L lysozyme solution (25 mg/mL) per mL cell precipitate, digested at 37 C. for 30 minutes, and then centrifugated at 10,000 rpm for 10 minutes. Nucleic acid electrophoresis was performed on the supernatants obtained by pTMSCA and pTrcHis-MS2 and comparison between the yield of the expressed products was conducted (FIG. 1).

[0047] {circle around (10)} Comparison results between the lysate supernatants of the expressed products after exogenous fragments of different length packaged by pTMSCA and pTrcHis-MS2: the plasmid pTrcHis-MS2 was respectively linked with an exogenous gene fragment of different length (fragment A (A<500 bp) and fragment B (500 bp<B<1000 bp)) via two restriction enzyme digestion cutting sites for KpnI and HindIII, and then subjected to transformation and sequencing to construct recombinant plasmids pTrcHis-MS2-A and pTrcHis-MS2-B. Referring to the construction method of pTrcHis-MS2-A and pTrcHis-MS2-B, the recombinant plasmids pTMSCA-C (1000 bp<C<1500 bp) and pTMSCA-D (D>1800 bp) were constructed respectively 4 recombinant strains were processed referring to step {circle around (9)}, and comparison between the yield of the expressed products was conducted (FIG. 2).

EXAMPLE 2

Construction of a Novel Virus-Like Particle Expression Vector pTMSCA2C

[0048] Using the plasmid pTMSCA as a template, the synthesized double-CP gene encoding MS2 bacteriophage coat protein in which two CP genes were linked in series was inserted between XhoI/HindIII restriction cutting sites by the method of restriction enzyme digestion and insertion to give the novel virus-like particle expression vector pTMSCA2C, wherein, the first CP gene sequence was the gene sequence coding wild type MS2bacteriophage coat protein with the deletion of the stop codon thereof; and the second CP gene sequence was the gene sequence coding coat protein on the vector pTrcMS (SEQ ID NO: 2), which constituted a series structure together with the first CP gene sequence. The specific construction method is as follows:

[0049] {circle around (1)} Using the plasmid pTMSCA as a starting vector, the gene coding sequences corresponding to the MS2 bacteriophage coat protein were modified according to the following mode: the first CP gene sequence was the gene sequence coding wild type MS2 bacteriophage coat protein with the deletion of the stop codon thereof was deleted; the second CP gene sequence was the gene sequence coding coat protein on the vector pTreMS; the 2CP gene sequence synthesized by Huada Gene Biology Co., Ltd. was inserted into the vector pUC57; and the obtained recombinant vector was named as pUC57-2CP.

[0050] {circle around (2)} The plasmids pUC57-2CP and pTMSCA were subjected to double restriction enzyme digestion with XhoI and HindIII respectively. The digestion system is as follows: XhoI/HindIII, 10 U; 10restriction enzyme digestion buffer, 5 L; plasmid template, 1 g; add ddH.sub.2O to 10 L. The reaction was performed at 37 C. for 2 hours. Respectively, the products of the restriction enzyme digestion were subjected to gel recovery and purification and named as pUC57 -2CP (X/H) and pTMSCA (X/H).

[0051] {circle around (3)} Using DNA ligation kit (Takara, Code No. 6022), the pUC57-2CP (X/H) and pTMSCA (X/H) were linked together. The reaction system and condition are as follows: pUC57-2CP (X/H)/pTMSCA (X/H), each 100 ng; solution I, 5 L, add ddH.sub.2O to 10 L. The ligation reaction was performed overnight at 16 C.

[0052] {circle around (4)} 2.5 L of the above-mentioned ligation product was taken and transformed into E. coli Competent Cell JM109 through heat-shock transformation. The obtained product was cultured overnight at 37 C. The positive clones were picked and identified by sequencing. The primers used for identification were: Primer-U1: 5-GACAATTAATCATCCGGCTCG-3 and Primer-L1: 5-GATCTTCGTTTAGGGCAAGGTAG-3.

[0053] The plasmid identified as having the correct sequence was named as pTMSCA2C.

[0054] {circle around (5)} Comparison results between lysate supernatants of the expressed products after exogenous fragments of different length packaged by pTMSCA2C, pTMSCA and pTrcMS (ZL201110445022.5): referring to step ED in Example 1, pTMSCA2C-D (D>1,800 bp), pTMSCA-D (D>1,800 bp) and pTrcMS-A (A<500bp) were constructed by using plasmids pTMSCA2C, pTMSC-AP and pTrcMS. Three recombinant strains were processed referring to step {circle around (9)} in Example 1, and comparison between the yield of the expressed products was conducted (FIG. 3).

EXAMPLE 3

Preparation of Schmallenberg (SB) Virus-Like Particles

[0055] 1. Construction of Plasmid pTMSCA2C-SBV

[0056] The plasmid pGEM-T-SBV and plasmid pTMSCA2C prepared in Example 2 were subjected to double restriction enzyme digestion with KpnI and HindIII and gel recovery and purification, respectively; and then linkage and sequencing were performed to construct the recombinant plasmid pTMSCA2C-SBV.

[0057] 2. Preparation of pTMSCA2C-SBV Virus-Like Particles

[0058] {circle around (1)} Expression and lysis: Specific method was carried out referring to step {circle around (9)} in Example 1.

[0059] {circle around (2)} Using Ni Sepharose 6 Fast Flow purification system (GE) the product was recovered from the supernatant of pTMSCA2C-SBV and the collected filtrate was the purified virus-like particle pTMSCA2C-SBV. The product was identified by SDS-PAGE protein electrophoresis.

[0060] {circle around (3)} RNA was extracted from the recovered virus-like particles and subjected to real-time PCR and RT-PCR to identify the purity of the obtained RNA solution. The primers used for identification were Primer-SBV-F: 5-TCAGATTGTCATGCCCCTTGC-3 and Primer-SBV-R: 5-TTCGGCCCCAGGTGCAAATC-3, respectively. Probe: 5-FAM-TTAAGGGATGCACCTGGGCCGATGGT-3. See SEQ ID NOs: 12-14.

[0061] 3. Morphological Identification of pTMSCA2C-SBV Virus-Like Particles

[0062] Firstly, the purified solution of virus-like particles was subjected to 1% uranyl acetate staining, then subjected to natural drying and finally subjected to morphological observation through a transmission electron microscope.

[0063] 4. Clinical Application of the pTMSCA2C- SBV Virus-Like Particles

[0064] Using purified SBV virus-like particle as a positive quality control product, SBV nucleic acid testing was performed on sheep serum clinical samples.

[0065] The purified SBV virus-like particles were detected by SDS-PAGE electrophoresis. Results were shown in FIG. 4. The target protein was located between 26 kDa and 48 kDa, the size of which was consistent with a size of 2 times of MS2 bacteriophage coat protein (27.4 kDa). Verification results of purity indicated that: for PCR identification, there was no amplification curve; for RT-PCR identification, there was a standard S amplification curve, suggesting that there were virus-like particles containing the SB target gene in the solution without DNA contamination (FIG. 5). Electron microscopy observation showed that polygonal particles with a diameter of about 26 nm, i.e. the expressed virus-like particles after induction, may be observed (FIG. 6). Clinical application tests indicated that after fluorescence quantitative detection, the nucleic acid detection of the positive quality control product of SBV virus-like particles showed a standard S curve, and the clinical serum samples were identified as negative by the nucleic acid detection, which was consistent with the serologic detection results.

[0066] Although the present invention is described in detail through the general description and specific embodiments above, modifications or improvements may be made based on the present invention, which is obvious to a person skilled in the art. Therefore, all of those modifications or improvements that are made without departing from the spirit of the present invention fall into the scope of the invention as claimed.

INDUSTRIAL APPLICABILITY

[0067] The present invention discloses a novel virus-like particle expression vector and a construction method thereof. When the virus-like particle is prepared by using the expression vector of the present invention, the yield and purity of the virus-like particle may be improved while the workload for preparation of virus-like particles may be greatly reduced.

REFERENCE DOCUMENTS

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[0069] [2] Talbot S J, Goodman S, Bates S R, et al. Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex. Nucleic Acids RES, 1990, 18(12):3521-3528.

[0070] [3] Qiuying E I, Yangjian C, Qiwei G, et al. Preparation of a chimeric Armored RNA as a versatile calibrator for multiple virus assays. Clin Chem,2006, 52(7):1446-1448.

[0071] [4] Yuxiang Wei, Changmei Yang, Baojun Wei, et al. RNase-Resistant virus-like particles containing long chimeric RNA sequences produced by two-plasmid coexpression system. Journal of clinical microbiology,2008, 46(5):1734-1740.