Cloning And Expressing Of E2Gene Of Bovine Viral Diarrhoea Mucosal Disease Virus And Preliminary Development Of Indirect ELISA

Bovine viral diarrhea/mucosal disease(BVD/MD) is an infectious disease caused byBovine viral diarrhea virus(BVDV) in cattle, which is characterized by fever, gastrointestinalmucosal erosions, ulcers, exfoliates, hemorrhagic inflammation, diarrhea, leukopenia,reproductive disorders. The disease occurs worldwide and causes considerable economiclosses in cattle farming. At present, the prevention and control of this disease depends on acombining strategy of taking animal quarantine, and immunization and elimination. Thedeveloped countries such as Europe and America take measures of quarantine andelimination-based, supplemented by immunization approach to prevent and control thedisease, which has achive great progress. Due to lack of commercial vaccines and detectionreagents, however, the disease is widespread in many areas of China. Therefore, developingBVD vaccines and detection reagents, improving BVD prevention system are of greatsignificance for the sustained and healthy development of cattle industry in China.In this study, E2gene of BVDV-2SW isolate was cloned and expressed in E. coli andyeast system, respectively. The reactogenicity of recombinant E2protein was analyzed bySDS-PAGE and Western blot. From all recombinant proteins, recombinant E2protein withhigh reactogenicity was selected as coating antigen to develop indirect ELISA for thedetection of BVDV antibodies, which laid a foundation for the development of diagnosticreagents for BVDV. The methods and main results are as follows:1. Cloning and prokaryotic expression of BVDV E2gene.The the total RNA wasextracted from cell infected by BVDV-2SW isolate, BVDV E2gene was amplified usingRT-PCR. Then, PCR products were digested by EcoR I and Not I, the recovered fragmentwas inserted into prokaryotic expression vector pET-28a(+) and pET-32a(+) to generaterecombinant pET-28a-E2and pET-32a-E2. pET-28a-E2and pET-32a-E2were transformedinto E.coli BL21(DE3), the recombinant protein was highly expressed under conditions whichwas kept for6hours in37℃and1.0mmol/L IPTG. SDS-PAGE revealed that the expressedprotein appear in expected size with a molecular mass of32kDa and44kDa. Western blotshowed that the recombinant E2protein can specifically react with anti-BVDV antibodies,confirming the recombinant E2protein has good reactogenicity.2. Expression of BVDV E2gene in Pichia pastoris.The E2gene of BVDV was amplifiedby RT-PCR. Products were digested with EcoR I and Not I, then inserted into the yeastexpression vector pPIC9K to generate recombinant plasmid pPIC9K-E2. After Sal I linearized,pPIC9K-E2was electroporatd into Pichia GS115. The recombinant P.pastorisGS115/pPIC9K-E2was screened by G418resistance and further confirmed by PCR, andrecombinant transformants were successfully obtained with G418resistance(1.0mg/mL) and the phenotype His+Mut+;SDS-PAGE showed that E2protein was successfully expressed witha relative molecular mass of23.2kDa, which is consistent with the that of theoretical analysis.Western blot showed that the recombinant E2protein can react with anti-BVDV antibodies,confirming the recombinant E2protein has good reactogenicity.3. The purification of recombinant E2protein and preliminary establishment of indirectELISA method.The recombinant E2with high reactogenicity was purified, and the purifiedprotein E2was used to develop indirect ELISA. Through the detection, the result showed thatthe developed indirect ELISAmethod with good specificity and sensitivity.