Ernst H, Stroup D
Department of Clinical Virology, James N. Gamble Institute of Medical Research, Cincinnati, OH 45219.
Gene. 1988 Sep 7;68(2):345-56. doi: 10.1016/0378-1119(88)90037-6.
The gene for the major inner capsid protein VP6 of human rotavirus strain Wa has been cloned and placed into a bacterial expression vector under the control of the inducible hybrid trp-lac (tac) promoter. Recombinant VP6 was produced at low levels in a cell-free Escherichia coli transcription-translation system programmed with this expression plasmid. The yield of VP6 synthesized in the extract could be increased several-fold by introduction of point mutations upstream and downstream from the start codon. Upon induction with IPTG, E. coli JM105 cells harboring the mutated expression plasmid produced VP6 as shown by immunoblotting of proteins from bacterial lysates with anti-Wa antiserum. Recombinant VP6 appeared to inhibit the growth of E. coli and did not accumulate in the cells to high levels. Conformational analysis with a monoclonal antibody suggested that bacterially produced VP6 adopted an oligomeric structure characteristic for native VP6.
人轮状病毒Wa株主要内衣壳蛋白VP6的基因已被克隆，并置于可诱导的trp-lac（tac）杂交启动子控制下的细菌表达载体中。用该表达质粒编程的无细胞大肠杆菌转录-翻译系统中，重组VP6的产量较低。通过在起始密码子上下游引入点突变，提取物中合成的VP6产量可提高几倍。用IPTG诱导后，携带突变表达质粒的大肠杆菌JM105细胞产生了VP6，用抗Wa抗血清对细菌裂解物中的蛋白质进行免疫印迹分析即可证明。重组VP6似乎抑制了大肠杆菌的生长，且未在细胞中高水平积累。用单克隆抗体进行的构象分析表明，细菌产生的VP6具有天然VP6特有的寡聚结构。
