Expression Of E2 Protein Of Bovine Viral Diarrhoea Virus In E.coli, Establishment And Application Of A Nest RT-PCR Method For Detection

Bovine viral diarrhea is a basic disease for cattle. It may cause bovine performance decrease and cause substantial ecnomic loss for bovine breeding. The genome of bovine viral diarrhea virus encodes eleven proteins. Among them glycoprotein E2 is one of the major protective antigens of virus. It may induce the production of neutralizing antibody, protecting animals from homologous virus challenge. The viral RNA was extracted from MDBK cells inoculated with bovine viral diarrhea virus BA strain. Based on published BVDV genome sequence, a pair of primers was designed using Oligo6 biology software for amplification of E2 gene, in which transmembrane domain and hydrophobic region of E2 protein was removed. A fragment of about 1000bp long was amplified by RT-PCR and cloned into pMD18-T-vector. Then recombinant vectors were identified with enzyme cutting and sequencing. Then the target fragment was directionally cloned into pET30a vector and the recombinant vector was transformed into E coli BL21 for expression. The transformed bacteria were cultivated and induced with IPTG then a recombinant protein was expressed in inclusion body forms. After denaturation, purification and renaturation, the recombinant protein was analyzed by western blotting and indirect enzyme-linked immunosorbent assay. The results showed the recombinant protein has a good immunogenicity. That is a better basis for diagnostic research on bovine viral diarrhea.Meanwhile the nested primers for amplification of BVDV were designed on the basis of 5'end consensus sequence of BVDV genome sequence which had been published in GenBank by using Oligo6 software.Two pairs of primers were designed. The outer primer will amplify an expected fragment of 294bp in length. The inner primers will amplify an expected fragment of 154bp in length. With blast analysis, the two pairs of primers may detect all kinds of BVDV, CSFV and BDV. Using this two pairs of primers, a nest RT-PCR detection method for BVDV was established. Using established BVDV RT-PCR detection method, various kinds of BVDV suspected materials and sera were detectd. The detected samples included bovine sera collected in cattle farms, commercial new-born calf sera and adult bovine sera for cell culture, miscarriage of foetus tissues. Nucleotide sequence analysis showed this method is specific for BVDV, CSFV RNA detection. The method is also sensitive, accurate and quickly, and may be used for BVDV and other pestivirus RNA detection. Based on nucleotide sequence analysis and phylogenetic tree analysis of amplified fragments, initial results showed that the domestic epidemic BVDV strain is BVDV I type, which includes 4 different subtypes. These results will provide a technical supporting for domestic BVDV prevention and control.This study established a BVDV RT-PCR diagnosis method, which can detect BVDV and CSFV. In the meantime, the truncated BVDV E2 gene was successfully cloned into pET30a expression vector.The recombinant E2 protein was expressed in inclusion body forms.After denaturation, purification and renaturation, the recombinant protein E2 still has a good immunogenicity. An initial investigation for BVDV in China was done by using established RT-PCR diagnosis method. A wishing to revoke attention of BVD in China was expected.