Expression Of NS3 Gene Of Classical Swine Fever Virus In E.coli And Preparation And Identification Of Its Monoclonal Antibodies

For studying on function of NS3 protein in CPE caused by CSFV and exploring the pathogenic mechanism of CSFV, it's necessary to develop the monoclonal antibodies against NS3 protein in order to examine expression difference of NS3 protein in CSFV-infected cells with and without CPE and expression situation of NS3 protein in swine cells transfected NS3 gene. Thus the relationship between NS3 protein and CPE could be presumed according to the extent of CPE or cell apoptosis. This research is momentous to explain the pathogenic mechanism of CPE caused by CSFV.This research consisted of two parts. In the first part, CSFV shimen strain NS3 protein was expressed in E.coli and purified through Ni-chelating affinity chromatography, then the purified NS3 protein was injected into Balb/c mice, thus the specific anti-NS3 antibodies were induced in mouse serum. The second part included fusion of splenic cells and SP2/0, screening and subcloning of hybridoma cells, and identification of hybridoma cells and the monoclonal antibodies.1. NS3 gene of CSFV Shimen strain was amplified by PCR and its serine protease functional domain was cloned into prokaryotic expression vector pET-32a. Then it was co-digested by the restriction enzymes Sacâ… and Hindâ…¢and sequenced. The results showed that NS3 gene inserting sites, direction and reading frame were all right, therefore the recombinant plasmid pET-NS3 was obtained successfully, which was subsequently transformed into E.coli Rosetta. The recombinant fusion proteins were highly expressed after IPTG inducement and the molecular weight was about 43 ku as expected. Western-blot showed that the recombinant protein could be recognized by CSF positive serum. The recombinant protein was purified through Ni-chelating affinity chromatography, and the purity was above 90% explained by SDS-PAGE gel scan analysis. The UV absorption determination indicated that the recombinant protein concentration was about 0.5 mg/mL. More over, Balb/c mice were injected with purified recombinant NS3 protein and induced specific anti-NS3 antibodies in mouse serum, which provided the precondition for the production of monoclonal antibodies against NS3. Meanwhile, the purified NS3 protein is diagnostic basis for distinguishing between wild strain infected and vaccine immune swines.2. Splenic cells of Balb/c injected via tail vein were hybridized with SP2/0 by cell fusion and the hybridoma cells were screened by ELISA and subcloned approach. Finally four hybridoma cells (2G7,2F11,5D2 and 6F11) stably secreting anti-NS3 monoclonal antibodies were obtained, and ascites were induced to produce monoclonal antibodies. Hybridoma cells and the monoclonal antibodies were identified ultimately. The results indicated that hybridoma cells were stably secreting antibodies after freezing and thawing and passage culture. The McAbs showed high titer to purified NS3 protein with various affinity and all the McAbs belonged to IgGl subgroup. Western-blot indicated that four strain McAbs presented high specificity to the recombinant NS3 protein expressed in the prokaryotic cells. The 6F11 strain McAb could bind to NS3 protein expressed in eukaryotic cells highly specifically. In the end, we produced anti-CSFV NS3 monoclonal antibodies successfully, which were foundation for the further study on function of NS3 protein in CPE caused by CSFV.