Prokaryotic Expression Of N Protein Of Porcine Reproductive And Respiratory Syndrome Virus In E. Coli And Development Of Monoclonal Antibodies Against N Protein

Porcine reproductive and respiratory syndrome (PRRS) can cause reproductive failure in sows and respiration disturbance in piglets. Its pathogen PRRSV is a member of the family Arteriviridae, Nidovirales. The genome of PRRSV is a positive sense,15 kb single strand of RNA encode RNA depending polymerase(RdRp) and 7 structure proteins GP2A?GP2b?GP3?GP4?GP5?M?N. N protein is the most effective antigen in these viral proteins. After infected by PRRSV, the body can produce antibodies against N protein more early and efficiently than other viral proteins. In recent studies, N protein has been used in diagnosing the disease. In our study, N protein was expressed in E.coli and 3 N-specific monoclonal antibodys were prepared. The main research contents was as following:1. Expression of N gene of PRRSV SY0608 in prokaryocyte system:In order to express the N gene of PRRSV SY0608, a pair of primers that have BamHI and Hind?sites were designed according to the sequence of PRRSV SY0608 strain. The N gene was amplified from recombinant plasmid pMD18T-11155-14254 containing the ORF7 gene of PRRSV by PCR using the primers, and cloned into a prokaryotic expression plasmid pRSET-A. The ligated product was transformed into E.coli DH5a and then the positive clone was picked. Then a recombinant plasmid named pRSET-N-SY was constructed and identified with restriction enzyme digestion and sequenced. The plasmid pRSET-N-SY was transformed into E.coli BL21 and induced by 0.6mM isopropyl-?-D-thiogalactoside (IPTG) at 37?for 4h. The result of SDS-PAGE of the bacteria revealed that a fusion protein HIS-N-SY with molecular weight of 17 kDa was expressed in E. coli. The purified HIS-N-SY protein showed a strong reaction with the PRRSV-positive sera in Western blot assay.2. Development of monoclonal antibodies against N protein:Six to eight-week-old BALB/c mice were hypodermically immunized three times at 2-week interval with 50ug to 100 ug of the purified HIS-N-SY protein or PRRSV SY0608 emulsified in Freund's complete (first injection) or incomplete adjuvant (second and third injections) respectively. The mice received a final injection of 50 ug of the protein or PRRSV in PBS 3 days before fusion. The splenocytes of the immunized mice were fused with murine myeloma cells SP2/0, and the rate of fusion was 80%-90%, the positive rate of the first screen was 40%-60%. After subcloning 3 times, three hybridoma clones which produced McAbs steadily were screened by ELISA, named 2H7,4A11 and 1G9. McAbs 2H7 belongs to IgG1 isotype, and its light chain belongs to?type. The result of IFA showed that they all reacted strongly with the PRRSV S1 and SY0608 strain infected Marc-145 cells. Meanwhile, western blot indicated that they reacted specialy with N protein of both strains. The antibody titers of 2H7,4A11 and 1G9 in the cell cultural supernatant were 1:1280,1:320 and 1:640 in ELISA, respectively, and the hybridoma 2H7 in the ascites were 1:128000. after passaged by 20 times, the hybridoma clones still secreted the antibody with the same titer they were passaged 5 and 10 times.In summary, in this experiment, the N protein of PRRSV SY0608 strain was expressed and three hybridoma clones which produce McAbs against the N protein steadily were prepared. These McAbs could be used for identification of PRRSV and studing the function of the N protein.