Determination Of The Complete Genome Of Endogenous Jaagsiekte Sheep Retrovirus And The Expression Of Env Protein

Endogenous retroviruses(ERVs)are Betaretrovirus,and their genes are ubiquitous in the genomes of a number of animals.Some endogenous retroviruses are closely related to the development of its host placenta.Up to now,at least 27 copies of endogenous retroviruses have been discovered in the sheep genome.These endogenous retroviruses are associated with their corresponding Jaagsiekte sheep retrovirus(JSRV).So it is called endogenous jaagsiekte sheep retrovirus(enJSRV).enJSRV plays an important role in the formation of sheep placent,and the enJSRV envelope protein(Env)can play a catalytic role in sheep chorionic trophoblast cell fusion process,but the mechanism of action of enJSRV envelope protein is not clear yet.In order to further understand the mechanism of enJSRV and envelop membrane proteins,this experiment screened the lung tissue BAC library of OPA(Ovine pulmonary adenocarcinoma,OPA)disease and cloned the entire genome sequence of enJSRV.Then this thesis performed the structural analysis of the env gene,and constructed a prokaryotic expression plasmid,and induced envelope protein expression.Primers were designed based on the endogenous jaagsiekte sheep retrovirus DNA sequence(AF152615)provided in GenBank.The experiment was used to amplify and clone the sequence through PCR.Afterward the cloned results were sequenced and analyzed by bioinformatics.Then,the env gene fragment added with BamH I and Hind III restriction sites was cloned into pET-32 a vector to construct an expression plasmid,and the plasmid was transformed into E.coli BL21(DE3)to induce envelope protein expression.Finally,the envelope protein was detected and identified by SDS-PAGE and Western blot.The envelope protein was purified by nickel column affinity chromatography.The results of this study indicate that the enJSRV sequence is 7382 bp,in which the env gene fragment is 1836 bp in length and the homology of enJSRV and enJSRV-23 is 97.3%.Software like PSORTII and DNAStarpredict that enJSRV envelope protein is mainly located in the cytoplasm,mitochondrial inner membrane and other locations,and there exist multiple epitopes.IPTG was induced into pET32-env E.coli BL21(DE3)at 37 °C,0.5 mmol/L.Eight hours later,envelope protein in the form of inclusion body with a molecular weight of approximately 88 kD was obtained.The experiment determined the entire enJSRV gene sequence and constructed the PET32a-Env recombinant plasmid expressing the envelope protein.It also expressed and purified the envelope protein.The experiment lays the foundation for the preparation of specific antibodies,and provides a theoretical reference for the in-depth discussion of the biological functions of envelope proteins.