The Difference Of Immunogenicity Between E0and E2Gene Inbovine Viral Diarrhea/Mucosal Disease Virus

Bovine viral diarrhea virus is one member of the Flaviviridae, Pestivirus. It’s a serious pathogenseriously endanger to the livestock’ health around the world. This disease can cause patient high fever，oraland nasal mucosal bleeding or mucus oversecretion，reduction of milk production even off production，pregnant cow abortion or stillbirth， fetal anomalies and other symptoms. BVDV has serologicalcross-reactions with CSFV and BDV. At present, there is no commercial vaccines to prevent the disease, itis urgent to develop on effective and ideal vaccine.The study consists of two parts:To construct the BVDV E2gene eukaryotic expression vector,identify its expression and immunityactivity,and therefore provide the basis for later development of vaccine.According to the encodingsequence of BVDV E2gene in GenBank（NVDL strain),appropriate primers specific to E2gene of BVDVwere designed.A target DNA fragment was amplified by PCR and then the amplicon was purified andcloned into pMD19-T.After identifying by sequencing,the exact DNA fragment was cloned into theeukaryotic expression vector pVAX1,which was named pVAX1-E2.And then both of them were transfectedinto293T cells with2000.Western-blot was used to confirm that the E2protein from pVAX1-E2wasefficiently expressed.Then immunized6-weeks mice with the recombinant DNA vaccine intramuscularinjection method,detect the neutralizing antibody of the mice after14days、28days and42days,do thelymphocyte transformation experiment after42days.To acquire the E0and E2protein of BVDV by expressing in prokaryotic system.In this study,thecomplete length of were amplified by PCR using a Pair of specific primers designed according to theoptimized sequences from GenBank,and the products were cloned into prokaryotic expression vector pETto obtain the prokaryotic expressed plasmid pET-28a-E0and pET-30a-E2.The target gene was thenexpressed in the E.col cells by induced with IPTG and the expression was optimized with a properinduction time and temperature.The good immunological activity to BVDV antibodies was proved bySDS-PAGE and Western-blot analysis..The healthy mice were immunized intramuscularly three times withpurified proteins.Subsequently, the levels of IgG and the proliferative reaction of lymphocytes weredetected by ELISA and MTT. The results showed that compared with the control groups mice,both ofproteins produced a significant difference and the E2group is better.