Molecular Cloning And Sequence Analysis Of Envelope Glycoprotein Gene E2 In Isolation Strain From Bovine Viral Diarrhea Virus Of Deer

BVD was a contagious disease that caused by BVDV,the key features of it were fever,cough and cow aborted or outputted featus anomalies.BVDV was the type species of Pestivirus,the virus had only one serological type.BVDV could infected many animals,especially hazardous to the economic ruminants as deers.Because the deers was the important economic animal,and it would cause momentous loss to the deers because of conomic BVDV not only causing deers aborting,dead foetus,weak foetus,featus anomalies and producting viremia son,but also holding back the foraging and ingesting,causeing secondary infection,and influencing developmentment of immature deers and performance of deers,even resulting in bulk death of deers.Besides there were not vaccine for deers,so it would bring about great economic damage once the disease breaking out.And then the study for BVDV appeared especially important.Respecting the feature of epidemic condition and hard preventing,the study selecting E2 gene as the investigation object made foundation for seting up diagnostic method and manufactureing vaccine for BVDV.CP BVDV strain was isolated from deer field in Changchun using MDBK cell.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 morphology,biological property and immunoassay were coincidence with typical strain;the result of cell virulence test was 10^-55.Using a pair of specigicil appraisement primer to identificating,there was a strap at 300bp;Using a pair of specigicil gene typing primer to identificating,there was a strap at about 500bp～750bp,the result proved that the isolated strain was BVDV 1.We used primer F1,F2,R1,R2 to amplify E2 gene.The PCR product was 1.4kb by examining the fragment by electrophoresis.After purification and insertion into pMD18-T vector,the recombinant plasmid were obtained.Then they were transfected E.coli Top10 and screened positive clones by blue or white plaques.The recombinant plasmids were extracted and the inserted fragments were identificated by electrophoresis.Through the homology analysis of the nucleotide and amino acid of the sequences about E2 gene,it indicate that the isolated strain has the best homology with 184 strain,and minimum with Shinara.The others is Osloss 92.8%,CV24 74.6%,SD-1 74.6%,NADL73.5%,SH9 72.7%,ZM72.5%,Deer-GB1 73%,S10 72.1%.The anaysis of nucleotide and amino acid sequence indicated that E2gene lenghs 1122bp,and coded 374 aa.There were 16 cyctein residue and 4 N-glycosylationsite in the isolated strain which was the same to 184 strain and Osloss strain.