Expression Of PRRSV GP5 Recombinant Protein In Different Types And Analysis Of Neutralizing Activity Of Their Antiserum

Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the most economica-lly significant diseases of swine and is characterized by reproductive failure in sows and respiratory distress in growing pigs.Neutralizing antibodies play a crucial role in resistance to PRRSV infection.GP5 protein,which as an important part of PRRSV capsule,is considered to be the main protein inducing neutralizing antibody,and that who is also applied to PRRSV gene engineering vaccine research.However,the subunit vaccine using GP5 as the target gene could not induce high neutralizing antibody,and the cross protection was poor.Therefore,through the efficient expression of GP5 protein in vitro,and analysis of its ability to induce neutralizing antibodies would make sense for exploring the role of GP5 in immune protection and its application in PRRSV prevention and control thus to provide a theoretical reference.In the first experiment,we used Marc-145 cells to proliferate the PRRSV strain VR2332,which was stored in the laboratory.The ORF5 gene was amplified by RT-PCR method,and then connected to PMD18-T vector and sequenced.According to the sequencing of ORF5,two pairs of primers were designed to amplify an incomplete ORF5 gene fragment which lack of Transmembrane domain(66aa-125aa)and signal peptide(1aa-31aa)by using SOE-PCR,and then the incomplete ORF5 gene fragment was cloned into a prokaryotic expression vector PET-32 a to construct the prokaryotic expression vector PET-ORF5 NC.The recombinant plasmid was transformed into E.coli BL21,and the recombinant protein with a size of about 31 KD was successfully expressed with IPTG inducing by SDS-PAGE.After optimizing the concentration and time of IPTG,we get a high expression of recombinant protein.The inclusion bodies protein was then purified and renaturation.Western blot was perfomed respectively with mouse anti His monoclonal antibody and PRRSV positive serum as primary antibody in detection of recombinant protein,the result show that recombinant protein was good reactionogenicity.In experiment two,according to the OFR5 gene fragment sequence which were obtained by experiment one,another two pairs of primers were designed.One of themwas ued to amplify a complete ORF5 gene fragment and another for an incomplete ORF5 gene fragment which lack of Transmembrane domain(66aa-125aa)and signal peptide(1aa-31aa)by using PCR.The two fragment are respectively cloned into the p Fast-HTA vector to obtain two recombinant vector that were then be transformed into DH10-Bac cells that contain baculovirus shuttle plasmid.Three antibiotics with blue white screening was used to obtain two kind of different recombinant shuttle vectors named Bacmid-ORF5 and Bacmid-ORF5 NC and then were respectively transfected into Sf9 cells to obtain two recombinant baculovirus.Sf9 cells were infected respectively by these two kind of recombinant baculovirus.The expression production were identified and analyzed by SDS-PAGE and Western blot,the result showed that Sf9 cells respectively expressed two kind of recombinant protein,and one cantain a compelete GP5 with the expected size of 22 KD,and another an incompelete GP5 which lack of Transmembrane domain and signal peptide with the expected size of 17 KD,the two are both in nonglycosylated form.Recombinant proteins were purified by Ni affinity chromatography to got a high purity.In experiment three,Kunming mice were immunized respectively with the recombinant protein for preparation of antiserum.The titer of antiserum were detected by Indirect enzyme linked immunosorbent assay(ELISA)with purified PRRSV and the corresponding recombinant protein as coating antigen;The results show that,when used the recombinant protein as coating antigen,the ELISA titer of prokaryotic truncated GP5 is 1:25600,and 1:12800 for Baculovirus GP5 antiserum and the same for Baculovirus truncated GP5 antiserum.when used PRRSV as coating antigen,the ELISA titer against prokaryotic truncated GP5 antiserum is 1:200,and 1:800 for Baculovirus GP5 antiserum and 1:1600 for Baculovirus truncated GP5 antiserum.Porcine alveolar macrophages(PAMs)were used for PRRSV neutralization assay,Real-time PCR were used the to detect PRRSV m RNA copy.The maximum dilution of serum would be the neutralization titer of antiserum with the virus blocking rate was higher than 70%.Neutralization assay results showed that the titer were all lower than 1:2,such suggest that none of the three antiserum were able to neutralize PRRSV infection to PAMs,and as However the control of the anti-PRRSV positive serum can neutralize the virus infection with a Neutralizing titer of 1:8.