Construction And Expression Of Prokaryotic Expression Plasmid PET-32a/exJSRV-env

Ovine pulmonary adenocarcinoma(OPA)is a natural infection lung tumor of sheep which disease is chronic,progressive and contagious caused by Jaagsiekte sheep retrovirus(JSRV).OPA has emerged in many countries in the world and epidemic all sheep industry developed countries.In China it epidemic Heilongjiang,Inner Mongolia,Xinjiang,and Shandong,Seriously affect the development of sheep industry worldwide.Therefore,the diagnosis of OPA has important scientific significance for controlling and understanding its distribution.Due to the absence of circulating antibody in OPA and the inability to establish a pathogenic culture system in vitro,conventional immunological methods,virus isolation and identification cannot be used for diagnosis.At present,the laboratory diagnosis of the disease mainly uses PCR and immunohistochemical methods.This experiment has performed basic research for the diagnosis of the disease by immunohistochemical methods.Firstly,primers were designed based on the sequence of Jaagsiekte envelope gene(Accession No.JQ837489.1)published in Gen Bank,and p EGFP-C1/ex JSRV-env plasmid(recombinant plasmid of ex JSRV-env and eukaryotic expression vector p EGFP-C1)was performed.After PCR,restriction enzyme digestion and sequencing,it was confirmed that the recombinant plasmid contains the complete ex JSRV-env gene;The ex JSRV-env gene was amplified using the p EGFP-C1/ex JSRV-env plasmid as a template to construct a prokaryotic expression plasmid as p ET-32a/ex JSRV-env,which was identified by double enzyme digestion and sequencing.The recombinant plasmid p ET-32a/ex JSRV-env was transformed into E.coli Transetta(DE3)and induced by IPTG.The concentration of IPTG-induced fusion protein expression was optimized.The soluble protein expression was analyzed by SDS-PAGE.Western Blot identified fusion proteins.The results showed that the p EGFP-C1/ex JSRV-env plasmid Showing two bands 5500 bp and 2000 bp after the double enzyme digestion.The sequence of the p EGFP-C1/ex JSRV-env plasmid was identical to that of the NCBI database,indicating that the plasmid contains the ex JSRV-env gene.Double digestion of the p ET-32a/ex JSRV-env plasmid showed bands of approximately 5900 bp and1862bp,and the sequence of the p ET-32a/ex JSRV-env plasmid was identical to that of the NCBI database,indicating the successful construction of p ET-32a/ex JSRV-env prokaryotic expression plasmid;The recombinant plasmid p ET-32a/ex JSRV-env was expressed in E.coli Transetta(DE3)with IPTG-induced expression of 89 k Da protein,and the optimal IPTG induction concentration was 0.5mmol/L.The SDS-PAGE electrophoresis showed that the fusion protein mainly appeared in the bacterial solution.The disrupted pellets were expressed as inclusion bodies;Western Blot identification demonstrated that IPTG induced expression of the protein.The protein can be used to prepare JSRV Env polyclonal antibody,which lays a foundation for the diagnosis of OPA by immunohistochemistry.