DU Fangyuan, Chen Dayong, Zhang Yufei, Sun Xiaolin, Guo Wenqing, Liu Shuying
College of Veterinary Medicine, Inner Mongolia Agricultural University, Huhhot 010018, China.
Inner Mongolia Sainuo Sheep Pasture Corporation, Hohhot 010040, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Sep;32(9):1188-92.
Objective To explore the influence of the exogenous Jaagsiekte sheep retrovious (exJSRV) envelope protein (Env) on NIH3T3 cell proliferation. Methods A recombinant plasmid pcDNA4/myc-His/exJSRV- env carrying exJSRV- env gene was constructed, and then the correctness of the recombinant plasmid was identified by PCR, restriction enzyme digestion and sequencing. The recombinant plasmid pcDNA4/myc-His/exJSRV- env was transiently transfected into NIH3T3 cells by Lipofectamine(TM) LTX. After the transfection of the recombinant plasmid, the expression of exJSRV- env was detected by reverse transcription PCR and Western blotting. The effect of Env on cell proliferation was investigated by CCK-8 assay and plate colony formation assay. Results The recombinant eukaryotic expression plasmid containing exJSRV- env was successfully constructed as identified by PCR, restriction enzyme identification and sequencing. After the recombinant plasmid was transiently transfected into NIH3T3 cells, reverse transcription PCR and Western blotting showed the expression of exJSRV- env , and Env promoted NIH3T3 cell proliferation significantly. Conclusion JSRV Env was expressed successfully in the NIH3T3 cells and promoted the proliferation of NIH3T3 cells.
目的 探讨外源性绵羊肺腺瘤反转录病毒（exJSRV）包膜蛋白（Env）对NIH3T3细胞增殖的影响。方法 构建携带exJSRV-env基因的重组质粒pcDNA4/myc-His/exJSRV-env，然后通过PCR、酶切及测序鉴定重组质粒的正确性。采用Lipofectamine(TM) LTX将重组质粒pcDNA4/myc-His/exJSRV-env瞬时转染至NIH3T3细胞。重组质粒转染后，通过逆转录PCR和蛋白质免疫印迹法检测exJSRV-env的表达。采用CCK-8法和平板克隆形成试验研究Env对细胞增殖的影响。结果 经PCR、酶切鉴定及测序证实成功构建了含exJSRV-env的重组真核表达质粒。重组质粒瞬时转染至NIH3T3细胞后，逆转录PCR和蛋白质免疫印迹法显示exJSRV-env表达，且Env显著促进NIH3T3细胞增殖。结论 JSRV Env在NIH3T3细胞中成功表达，并促进NIH3T3细胞增殖。
