Construction and Prokaryotic Expression of the Fusion Gene Prrsv Gp5 and Mycobacterium Bovis Hsp70 the Fusion Expressing Plasmid Pet32-gp5-hsp70 Was Constructed and Expressed in Escherichia Coli Bl21. Ni

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease that has devastated the swine industry worldwide. Vaccination with live attenuated vaccine or inactivated vaccine is the main treatment to control PRRS. However, the disadvantages such as virulence resumption of the attenuated vaccine and low immunogenicity of the inactivated vaccine call for a more efficient and safer genetically engineered vaccine. In this study, the structural protein GP5 of the PRRS virus (PRRSV), one of the major protective antigens which stimulates a protective immune response was selected to develop a genetically engineered subunit vaccine. In order to promote the immune reaction of the host to GP5, heat shock protein 70 (Hsp70) was selected as immuno-adjuvant to enhance PRRSV GP5 immunogenicity. 2+-chelating resin was used to purify the His-tagged fusion protein expressed under optimized expressing conditions. The rabbit anti-GP5-Hsp70 fusion protein antibody was made, and Western blot assay verified the successful expression of the fusion protein, making it possible for further investigation whether Hsp70 could improve the immunogenicity of the PRRSV GP5 subunit vaccine, or evaluating the immunogenicity of the GP5-Hsp70 subunit vaccine.


INTRODUCTION
Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), a positive-sense single stranded enveloped RNA virus of the family Arteriviridae and the genus Arterivirus (Meulenberg 2000).PRRS has caused enormous financial losses to swine industry worldwide with clinical signs such as respiratory disease in growing pigs and reproductive failure in breeding swine
PRRSV genome is approximately 15 kb in size and consists of nine open reading frames (ORFs), with each ORF partially overlapping with consecutive ORF, and performs its own biological function, respectively (Conzelmann et al., 1993;Thiel et al., 1993).ORF5 is an important structural protein gene, encoding the major envelope glycoside protein GP5.GP5 is extensively considered to be the binding protein of the virus receptor, and is one of the most important antigen proteins eliciting antibody response in PRRSV infection, so the GP5 gene is a good candidate for developing genetically engineered subunit vaccines (Pirzadeh and Dea, 1998).However, the GP5 protein itself has poor immunogenicity (Foss et al., 2002).
Heat shock proteins (Hsps) are a group of highly conserved proteins, which are classified into Hsp90, Hsp70, Hsp60 and small Hsp families based on molecular size (Itoh et al., 1999).They play a significant role in protecting cells against various cellular stresses, such as heat, hypoxia, irradiation, oxidative stress and so on (Schroder et al., 2003;Atalay et al., 2009).They participate in antigen processing, presentation and T cell activation in immune response process, stimulate a specific cytotoxic T lymphocyte (CTL) response, and contribute to tumor immunity, infection immunity and the pathology mechanisms of autoimmune diseases (Koets et al., 1999;Wong, 1999;Atay et al., 2009;Tsan and Gao, 2009).They are involved in protein translocation, new protein assembly and the prevention of protein aggregation (Mokrushin et al. 2004).Hsps, expressed by prokaryotes and eukaryotes and their action as molecular chaperones have brought more focus on their important biological functions ( Guerra et al., 2005;Jonak et al., 2009).Among all Hsps, Hsp70 is the most conserved and widely studied.Hsp70 is mainly in the cytoplasm but also occurs in many cellular compartments (Itoh et al. 1999).
There have been many reports on Hsp70 acting as potent immuno-adjuvant to enhance both innate and adaptive immune system (Prohaszka et al., 2002), and not only a good immuno-adjuvant but also a protective antigen (Li et al., 2009;Tsan and Gao, 2009).Therefore, Hsp70, performing as an immuno-adjuvant, gives hopes for development of an efficient and safe PRRSV geneticengineering subunit vaccine (Ye et al., 2004).
The objective of this study was to construct a fusion vector with the PRRSV GP5 gene and the Mycobacterium bovis Hsp70 gene in pET-32a(+) plasmid, express the recombinant plasmid in Escherichia coli BL21, immunize the rabbit with the expressed fusion protein to prepare antiserum and detect the fusion protein to analyze its immunogenicity.

Virus, bacteria and plasmid
The PRRSV SCQ strain was isolated, identified and maintained by the Animal Biotechnological Center, Sichuan Agricultural University, China.E. coli DH5α and BL21, pMD18-T Vector were purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China).The attenuated strain of Mycobacterium bovis (M.bovis) was obtained from Disease Control Center of Ya'an City, Sichuan province, China.pET-32a(+) plasmid was maintained by Animal Biotechnological Center, Sichuan Agricultural University, China.

Primer design and sequencing
Two pairs of primers were designed based on the ORF of Hsp70 gene of the Mycobacterium tuberculosis (GenBank ID:BX248335) and GP5 gene (GenBank ID:DQ379479) of the PRRSV SCQ Strain.The upper (P1) and lower primer (P2) of GP5 gene included BamH Ⅰand Hind III enzyme digestion sites, respectively.In order to fuse Hsp70 gene at the end of GP5 gene, the termination codon of GP5 gene was deleted in the lower primer.The upper (P3) and lower primer (P4) of Hsp70 gene included HindIII and XhoI restricttion enzyme digestion sites, respectively (Table 1).Primer synthesis and the sequencing of the target genes were executed by Shanghai Invitrogen Biotech Company, China.

Amplification of target gene Hsp70 and construction of pMD18 -Hsp70 plasmid
The genome DNA of M. bovis was extracted using Myco DNAout Kit (TIANDZ GENE Company) as template.The full length of Hsp70 gene of M. bovis was amplified by PCR with P3/ P4 primers.PCR amplification of Hsp70 gene was carried out in a reaction mixture of 25 μl including 10.5 μl sterilized water, 12.5 μl 2×Taq PCR Master Mixture, 0.5 μl (20 μmol/l) of each primer (P3 and P4) and 1 μl template DNA.The thermal conditions and cycles were as follows: one cycle at 94°C for 5 min, followed by 30 cycles at 95°C for 1 min, 56°C for 40 s and 72°C for 2 min, and a final extension at 72°C for 10 min.The PCR product was inserted into pMD18-T vector to construct pMD18-Hsp70 plasmid.The pMD18-Hsp70 plasmid was transformed into DH5α host bacteria and extracted from the bacteria, then identified by PCR with primers P3/P4 and by doubledigestion with restriction enzymes HindIII and XhoI, and also by sequencing of Hsp70 gene in the plasmid.The pMD18-Hsp70 plasmid was kept at -20°C for use.

Amplification of target gene PRRSV GP5 and construction of pMD18-GP5 plasmid
Virus RNA was extracted from PRRSV SCQ strain using Trizol® Reagant (GIBCO BRL Company) and purified with High Pure RNA Isolation Kit (Boehringer Mannheim Corporation).Reverse transcription (RT) was conducted with random primers using reverse transcriptase kit (TaKaRa Biotechnology Co. Ltd., Dalian, China), and PCR was carried out in a reaction mixture as above except for different primers (P1and P2) and cDNA template.The thermal conditions were as follows: one cycle at 94°C for 5 min, followed by 30 cycles at 95°C for 30 s, 55°C for 40 s and 72°C for 30 s, and a final extension at 72°C for 10 min.
PCR product was detected and purified.The recombinant pMD18-GP5 plasmid was identified by PCR with primers P1/P2, double-digestion with restriction enzymes BamHI and HindIII, and also by sequencing.

Construction of fusion expressed plasmid pET32-GP5-Hsp70
pMD18-GP5 and pET-32(α+) plasmids were both double-digested by BamHI and HindIII and purified.The GP5 gene was inserted into the pET-32(α+) plasmid and transformed into DH5α host bacteria.The transformed DH5α bacteria were cultured and the recombinant plasmid was identified by PCR and double-digestion.The recombinant plasmid was named as pET32-GP5, and kept at -20°C until use.

Expression, purification and analysis of fusion protein GP5-Hsp70
The fusion gene GP5-Hsp70 was expressed following the conventional protocol to obtain the fusion protein GP5-Hsp70.Briefly, an overnight culture of BL21 containing the recombinant plasmid pET-32-GP5-Hsp70 was refreshed with LB broth containing 50 mg/L ampicillin, when the culture grew to an optical density (OD) of 0.6 at 600 nm, the cells were induced with 1 mmol/L isopropyl-β-Dthiogalactoside (IPTG) for 4 h.Then, the cells were harvested by centrifugation at 13,000 rpm for 1 min at 4°C.The cell sediment was collected and resuspended with sterilized H 2 O and equal volume of 2×SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) buffer, and SDS-PAGE was performed to detect the fusion protein.Different induction time, IPTG concentration and induction temperature were compared to study the optimal expressing conditions.
To detect the solubility of the fusion protein GP5-Hsp70, BL21 containing the recombinant plasmid pET32-GP5-Hsp70 was induced by IPTG following the optimal expressing conditions as optimized above.Cells were harvested by centrifugation at 13,000 rpm for 1 min at 4°C, resuspended in 20 mmol/l Tris-HCl (pH 8.0), antalzyme was added with the concentration of 1 mg into 1 ml BL21 solution, incubated at 37°C for 30 min, conserved at -20°C, frozen thawed after consolidation and repeated twice.The mixture was broken by ultrasonic wave on ice.The total lysate was divided into soluble and insoluble fractions by centrifugation at 13,000 rpm for 5 min at 4°C.The soluble and insoluble fractions were analyzed by 12% SDS-PAGE, and visualized using Coomassie Brilliant Blue.
Ni 2+ chelating resin (200 μl), equilibrated with 5 ml of sterile deionized H 2 O, 2 ml of 50 mmol/l Na 2 SO 4 , and 5 ml of PBS (pH 7.2), was used to purify protein from the expressing cells broken by ultrasonic wave with the conventional protocol mentioned above.The supernatant was passed through the column, followed by washing the column with 5 ml PBS (pH 7.2) and 5 ml wash buffer (20 mmol/l imidazole in PBS), respectively.The target protein was then eluted with 2 ml of elution buffer (200 mmol/l imidazole in PBS).The eluted fractions were detected by 12% SDS-PAGE.

Preparation of rabbit anti-GP5-Hsp70 antibody and Western blot analysis of protein GP5-Hsp70
The purified fusion protein GP5-Hsp70 was used to immunize white rabbits.All animal manipulations were approved by Sichuan Province Institutional Animal Care and Use Committee.The rabbits were immunized with 0.75 mg GP5-Hsp70 protein in complete Freund's adjuvant (Kothari et al., 2006;Liang et al., 2009).The booster immunization were performed weekly for 3 times after the first vaccination with 0.75 mg GP5-Hsp70 protein in incomplete Freund's adjuvant, then rabbit sera were collected at 10 days after the last immunization.The sera were tested by Western blot using the purified fusion protein GP5-Hsp70 as antigen (Done and Paton, 1995).
Western blot was carried out according to the standard procedure.Protein GP5-Hsp70 was separated by SDS-PAGE with 12% gel and electrophoretically transferred to a nitrocellulose membrane.The nitrocellulose membrane was then blocked overnight at 4°C with 10% skimmed milk in TBS (Tris buffered saline with 0.1%Tween 20, pH 8.0).After washing with TBS, the membrane was incubated for 60 min with the rabbit anti fusion protein GP5-Hsp70 antiserum at 4°C.The membrane was washed and incubated for 60 min with horseradish peroxidase-conjugated goat anti-rabbit antibody.After further washing, bands were visualized using diaminobenzidin (DAB) colour developing reagents.

Construction of the pMD18-Hsp70 plasmid
The Hsp70 gene PCR fragment was approximately 2000 bp (Figure 1A), coinciding with its theoretical size.The results of pMD18-Hsp70 positive recombinant plasmid PCR amplification and double-digestion with restriction enzymes HindIII /Xhol are shown in Figure 1B.The sequence of PCR fragment was 98.6% identity with the sequence of M. bovis in the GenBank.The pMD18-Hsp70 plasmid was successfully constructed.

Construction of pMD18-GP5 plasmid
The acquired PRRSV SCQ GP5 gene was approximately 530 bp (Figure 2A), coinciding with its theoretical size.The pMD18-GP5 plasmid PCR amplified from recombinant bacteria with primer pairs P1/P2 showed a single objective product of about 530 bp in site (Figure 2B), and the pMD18-GP5 plasmid double-digested with restriction enzymes BamHI and HindIII showed the objective band at 530 bp site as expected (Figure 2C).The sequence of GP5 PCR fragment was 99.7% identity with the sequence of PRRSV SCQ GP5 in the GenBank.The GP5 gene was successfully cloned into pMD18-T vector.

Expression of fusion protein GP5-Hsp70
The recombinant plasmid pET32-GP5-Hsp70 was expressed in host bacteria E. coli BL21.SDS-PAGE result showed that the molecular weight of the target Histagged fusion protein was about 110 kDa (containing 6×histidine tag sequences), which was consistent with the theoretical value (Figure 4A).Several expression conditions were experimented, when the cell density for pET32-GP5-Hsp70 induction was at OD 600 of 0.6, and the optimal protein expression occurred at 4 h post-induction with 0.5 mmol/l IPTG at 30°C (Figure 4B, C and D), and the fusion protein appeared mainly in the pellet of the induced pET32-GP5-Hsp70 transformed bacteria (Figure 4E).

Purification and Western blot assay of fusion protein GP5-Hsp70
Ni 2+ -resin purified his-tagged GP5-Hsp70 protein was detected at 110 kD site by SDS-PAGE showing a single objective band (Figure 5A).The titer of rabbit anti-GP5-Hsp70 antibody was 1:2 5 when the antibody was serial diluted and detected with agarose gel double diffusion method.When the purified fusion protein reacted with rabbit anti-GP5-Hsp70 antibody by Western blot, the antibody recognized the GP5-Hsp70 fusion protein at the site of appropriate molecular weight (Figure 5B).PRRS is difficult to be controlled.The best way to control PRRS is to apply effective vaccine.In this study, we successfully constructed and prokaryotic-expressed the fusion gene PRRSV GP5 and M. bovis Hsp70.The purified fusion protein was used to immunize the rabbit and produced specific antibody confirming the development of effective subunit vaccine of PRRS.
Poor immunogenicity and weak cross protection of PRRSV vaccines are the main issues in PRRSV immunezation.The utilization of vaccine adjuvants could improve vaccine immunogenicity (Royaee et al., 2004;Li et al., 2009).Hsp70 has attracted much interest for its potential in enhancing immunogenicity (Li et al., 2007;Zhang et al., 2007).In this study, we successfully fused the most important PRRSV antigen coding gene GP5 with Hsp70 gene from M. bovis, constructed and expressed fusion protein GP5-Hsp70.The efficiency of fusion protein GP5-Hsp70 in enhancing immunogenicity, cell mediated immunity and antibody responses should be further studied.

Figure 1 .
Figure 1.PCR amplification of M. bovis Hsp70 gene and identification of the recombination vector pMD18-Hsp70.A. Result of PCR amplification for M. bovis Hsp70 gene.Lane M, DNA marker 2,000; Lane 1, the amplified product of M. bovis Hsp70 (about 2,000 bp).B. Identification of the recombination vector pMD18-Hsp70 by PCR and restriction enzymes digestion.Lane M, DNA marker 4,500; lane 1, the amplified product of M. bovis Hsp70 (about 2,000bp) from the recombinant plasmid pMD18-Hsp70; lane 2, the recombinant plasmid pMD18-Hsp70 digested with HindIII and XhoI (the PCR products with about 2,000 bp and the pMD18-T vector of about 2,700 bp)

Figure 2 .
Figure 2. PCR amplification of PRRSV GP5 gene and identification of the recombination vector pMD18-Hsp70.A. Result of PCR amplification for PRRSV GP5 gene.Lane M, DNA marker 4,500; lane 1, the amplified product of PRRSV GP5 (about 530 bp); B.Identification of the recombinant bacteria by PCR.lane M, DNA marker 4,500; lane 1, the false amplified product of PRRSV GP5 from one selected recombinant bacteria; lanes 2, 3 and 4, the correct amplified product of PRRSV GP5 from three selected recombinant bacteria (the PCR products with about 530 bp); C. Identification of the recombination vector pMD18-GP5 by restriction enzymes digestion.Lane M, DNA marker 4,500; lane 1, the recombinant plasmid pMD18-GP5 was digested with BamHI and HindIII (the PCR products with about 530 bp and the pMD18-T vector about 2,700 bp).

Table 1 .
The primers of PRRSV GP5 gene and Hsp70 gene of M. bovis.