Molecular Cloning And Sequence Identification Of The Envelope Glycoprotein Gene E₂ In NADL Strain Of Bovine Viral Diarrhea Virus

Bovine Viral Diarrhea Virus (BVDV),a pestivirus ,is the causative agent of bovine viral diarrhea -mucosal disease in cattle throughout the world . Besides clinically observed diarrhea , it frequently causes fever ,oral ,ulceration,caugh ,leukemia and persistent infection in some cases . The genome of BVDV consists of a single stranded RNA of positive polarity ; which encodes all the structual proteins and replication components .The front part of the genome translates one polyprotein and processes to Eo(gp48),E1(gp25),E2(gp53)respectively . Along these structural proteins , the E2 protein is known to be a major envelope protein related to neutralizing activity in vivo . So the molecular cloning and sequence identification of BVDV E2 gene has important biological significance which has it convenient to be used .Bovine viral diarrhea virus were propagated by MDBK cells . After five to six continuously and rapidly passing, the cells emerged evident cytopathic effect .Then the infected cells and its solution were harvested . After freezing and thrawing three times , the virus is concentrated by ultracentrifugation.The BVDV NADL strain is cytopathogenic . Searching fromGenBank several CP strains' sequence of E2 gene about Deer-N21 ,SH9, NewYork-I and so on were found . According to the homologous sequence they were designed and synthesised a pair of primers by the biology software Primer 5 and added Bal I and Nco I site to the 5 ' end which can be used for the application of E2 gene and subcloning .During extracting the RNA genome and retranscripting of the E2 gene , more attention was paid on every materials for DEPC processed . The regents which was used were compounded with DEPC water . The E2 gene was amplified by RT-PCR , then examined the fragment by electrophoresis . After purification and insertion into pUCm-T vecter , the recombinant plasmid pBNE2PI and pBNE2PII were obtained . Then they were transfected E .coli JM109 and screened positive clones by blue or white plaques .The recombinant plasmids were extracted and the inserted fragments were identificated by electrophoresis . Through the analysis of the nucleotide and ammo acid of the sequences about E2 gene ,it express that the E2 gene includes about 1100bp which encodes about 370 amino acides .It builds a certain methods on biological study of E2 gene by this expriment through designing primer meticulously and cloning of envelope glycoprotein gene E2 .