The Study On The NIH3T3 Cells Transfected With PcDNA4/myc-His/exJSRV-env

Ovine pulmonary adenomatosis (OPA) was caused by Jaagsiekte sheep retrovirus (JSRV), induced chronic, contact, infectious and neoplastic diseases. JSRV has the similar structure and genetic organization with other retroviruses. The genome contains the gag genes, which encodes the structural proteins of the virus capsid; pro encodes the aspartic protease (PR), pol encodes the reverse transcriptase (RT), and integrase (IN); and env, which encodes the envelope glycoproteins (Env) present on the surface of the virus particle. The envelope gene is mainly responsible for the specific binding for virion and the target cells meanwhile inducing the membrane fusion to make virus particles enter into the target cells. The envelope protein is the major oncogenic protein.To further explore the function of exJSRV envelope protein and reveal the ca-rcinogenic mechanisms of JSRV. This experiment built the exISRV-env recombina-nt plasmid pcDNA4/myc-His/exJSRV-env, exogenous Jaagsiekte sheep retrovirus e-nvelope genes were amplified from the sheep lung tissue of Inner Mongolia natur-ally infected sheep pulmonary adenoma, identification accuracy by PCR, enzyme d-igestion and nucleic acid sequencing methods. The recombinant plasmids pcDNA-4/myc-his/exJSRV-env were transiently transfected into NIH3T3 cells by Lipofecta-mine LTX. After the transfection of the recombinant plasmid, the exJSRV-env gen-es were detected by RT-PCR and the envelope protein was detected by Western bl-ot. The effect of Env on cell proliferation was investigated by CCK8 method and p-late colony formation experiment. Results showed that the experiment successfully constructed recombinant eukaryotic expression plasmid pcDNA4/myc-His/exJSRV-en-v, after indentified by PCR, restriction enzyme identification and sequencing. The re-combinant plasmid transient transfected into the NIH3T3 cells, then detected the exp-ression of envelope protein. CCK-8 results showed that the proliferation activity of the cells which transfected with pcDNA4/myc-His/exJSRV-env was significantly hi-gher than the control (blank and negative) cells (P<0.01); The difference between the cells transfected with the empty plasmid pcDNA4/myc-His and the non transfe-cted cells was not significant (P> 0.05). Colony experimental results showed that: by SPSS software analysis, the clone formation rate of the cells transfected with pcDNA4/myc-His/exJSRV-env was significantly higher than that of the pcDNA4/m-yc-His transfectrd cells and the non-transfected cells (P<0.05), there was no signi-ficant difference between the cells transfected with pcDNA4/myc-His empty plasm-id cells and non-transfected cells (P> 0.05).The expression of EGFR was detected by Western blot, analyses grayscale by Image J, the result shows that the expressi-on of EGFR of the cells which transfected with pcDNA4/myc-His/exJSRV-env was significantly higher than control cells (P< 0.01); The difference between the cells transfected with the empty plasmid pcDNA4/myc-His and the non transfected cells was not significant (P> 0.05).This study showed that envelope protein of Jaagsiekte sheep retrovirus could promote the proliferation and colony formation of NIH3T3 cells, and it also could promote the expression of EGFR. It provided an experimental basis for further exploring the oncogenic mechanisms of Jaagsiekte sheep retroviral envelope protein.