VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells.
VSV-G Viral Envelope Glycoprotein Prepared from Pichia pastoris Enhances Transfection of DNA into Animal Cells.
复制标题
DOI：
10.4014/jmb.1611.11082
复制
发表时间：
2017-06
影响因子：
2.8
通讯作者：
Xin Liu;Ying Dong;Jingquan Wang;Long Li;Zhen-Min Zhong;Yun-pan Li;Shao-jun Chen;Yu-cai Fu;Wen-can Xu;Chi-ju Wei
中科院分区：
文献类型：
--
作者：
Xin Liu;Ying Dong;Jingquan Wang;Long Li;Zhen-Min Zhong;Yun-pan Li;Shao-jun Chen;Yu-cai Fu;Wen-can Xu;Chi-ju Wei
水泡性口炎病毒G糖蛋白（VSV-G）已被广泛用于假型逆转录病毒，慢病毒和人工病毒载体。本研究的目的是建立一个潜在的大规模生产VSV-G的方法。为此，将VSV-G与N-末端His-标签一起克隆到毕赤酵母表达载体pPIC 3.5K中。通过PCR鉴定含有VSV-G表达盒的三个克隆（Mut）。所有克隆在扩增培养基中正常增殖，而在诱导条件下增殖显著降低。Western blot检测到VSV-G蛋白在细胞裂解液中的表达，并在诱导后96 h观察到最高表达水平。VSV-G也可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Ad 293细胞中，并能够诱导细胞融合，导致胞浆蛋白的转移。最后，通过流式细胞术和荧光素酶测定来分析VSV-G介导的DNA转染。将VSV-G裂解物与pGL 3-对照DNA复合物一起孵育使Ad 293和HeLa细胞中的荧光素酶活性增加约3倍。同样地，VSV-G裂解物与pCMV-DsRed DNA复合物的温育将转染到Ad 293中的效率提高了10%，并且将转染到HeLa细胞中的效率提高了约1倍。总之，这些结果表明，VSV-G可以从具有生物功能性的巴斯德毕赤酵母中产生，表明大规模生产病毒糖蛋白是可行的。
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 Pichia pastoris expression vector pPIC3.5K. Three clones (Muts) 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 P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.