Construction Of A Cell Line Stably Expressing BVDV-NS5B

Bovine viral diarrhea(also known as mucosal disease)is one of the infectious diseases that must be reported by the Office International des épizooties(OIE).The pathogen is Bovine viral diarrhoea virus(BVDV).BVDV is a representative member of the genus Phaviviridae Pestivirus.BVDV can infect animals mainly including cattle,sheep and pigs,and it has recently been found that BVDV can also infect mice.However,its natural host is cattle,especially young cattle are the most susceptible.After infection with BVDV,the symptoms of cattle are caused,and the symptoms are different.Some of them are acute infections.Some animal with persistent infections(PI)have no obvious symptoms,but become the main source of infection.Due to its immunosuppressive effect,BVDV also causes secondary diseases to amplify its harm.At present,in the field of the prevention and control of BVDV,European countries and American have rich experience,mainly adopting two purification strategies of “screening +elimination” and “immunization + elimination”.The former is based on effective diagnosis and has high elimination cost;the latter relies on efficient vaccine immunization and is suitable for areas with high density of cattle and high prevalence of BVDV.Therefore,the second strategy is more suitable for the current situation in China.To control the popularity of BVDV in China,research and production of high-efficiency vaccines has become a top priority.The BVDV genome is positive-stranded RNA with approximately 12,500 nucleotides,and an open reading frame encodes a large protein that is then cleaved into 12 proteins.The last protein is its RNA polymerase NS5 B.The genome lacking this protein will not replicate,and the virus will not proliferate in the host cell,losing the ability to continuously infect.Our laboratory has successfully constructed the NS5B-deficient BVDV cDNA,so a production cell line stably expressing the NS5 B gene was constructed,and the NS5B-deficient BVDV vaccine virus was propagated in a matched cell line in a genetically complementary manner.The completion of theproject will be expected to make up for the shortcomings in the production of BVDV vaccine in China,which is of great significance for controlling BVDV in China.The upstream and downstream primers were designed and synthesized according to the NS5 B nucleic acid sequence,and the NS5 B nucleic acid sequence was amplified by PCR using the cDNA which was reverse-transcribed from the RNA extracted from the BVDV virus solution in the laboratory as a template,and then the pLVX-IRES-Puro vector was used.The pLVX-IRES-Puro-HA-NS5 B recombinant plasmid was constructed by ligation.After the recombinant plasmid was identified,293 T cells were co-transfected with packaging vectors(PLP1,PLP2,pLP/VSVG),and virus-infected MDBK cells were collected at 48 hours and 72 hours,respectively.After 48 hours of infection,the medium containing puromycin was screened.Positive cells,until the emergence of monoclonal and expanded culture.In addition,pLVX-IRES-Puro-HA-NS5 B was directly transfected into BHK cells,and a BHK cell line expressing NS5 B protein was obtained by puromycin screening.In this study,the NS5 B epitope was designed and successfully expressed by prokaryotic expression system and purified.The highest concentration of expression was determined to be430 ?g/mL,and then the mice were emulsified by Freund's adjuvant.After immunization three times,the eyeball was removed and the antibody of NS5 B was obtained.The serum of NS5 B was determined by ELISA titer and the highest titer was 1:64000.Therefore,effective serum was obtained for the late Western Blot to detect the cell line expressing NS5 B protein.In conclusion,this study successfully obtained anti-BVDV-NS5 B serum,successfully constructed BVDV-NS5 B stably expressed BHK cell line,provided a matching production cell line for NS5B-deficient BVDV vaccine virus,and laid a BVDV gene deletion virus for reverse complementation.