Liu Xin, Dong Ying, Wang Jingquan, Li Long, Zhong Zhenmin, Li Yun-Pan, Chen Shao-Jun, Fu Yu-Cai, Xu Wen-Can, Wei Chi-Ju
Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China.
Laboratory of Cell Senescence, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China.
J Microbiol Biotechnol. 2017 Jun 28;27(6):1098-1105. doi: 10.4014/jmb.1611.11082.
Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into expression vector pPIC3.5K. Three clones (Mut) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.
水泡性口炎病毒G糖蛋白（VSV-G）已被广泛用于逆转录病毒、慢病毒和人工病毒载体的假型化。本研究的目的是建立一种大规模生产VSV-G的潜在方法。为此，将带有N端His标签的VSV-G克隆到表达载体pPIC3.5K中。通过PCR鉴定出三个含有VSV-G表达盒的克隆（Mut）。所有克隆在扩增培养基中均能正常增殖，而在诱导条件下增殖显著降低。通过蛋白质印迹分析在细胞裂解物中检测到VSV-G蛋白，诱导后96小时观察到最高表达水平。VSV-G也可以从酵母原生质体的条件培养基中获得。此外，VSV-G可以整合到Ad293细胞中，并能够诱导细胞融合，导致细胞质蛋白的转移。最后，通过流式细胞术和荧光素酶测量对VSV-G介导的DNA转染进行了检测。将VSV-G裂解物与pGL3-control DNA复合物孵育可使Ad293和HeLa细胞中的荧光素酶活性提高约3倍。同样，将VSV-G裂解物与pCMV-DsRed DNA复合物孵育可使Ad293细胞的转染效率提高10%，使HeLa细胞的转染效率提高约1倍。总之，这些结果表明VSV-G可以通过生物功能产生，证明了大规模生产这种病毒糖蛋白是可行的。
