Construction and effective expression of the prokaryotic expression vector containing BHV-1 gB gene
2007
Wu Chuntao | Wang Jianhua | Hou Xiangshan
English. The gB gene fragment of bovine herpesvirus-1 Bartha Nu/67 strain was amplified by PCR and inserted into prokaryotic expressing vector pBAD/TOPO. The recombined plasmids were then transformed into E. coli TOP10 for expression. The expression was optimized with proper inducing conditions of 0.2 g/L L-Arabinose and 5 hours induction. The SDS-PAGE assay showed that the recombined plasmids could be expressed in inclusion body at a high level when induced with L-Arabinose.The purity of fusion proteins which was purified by magnehis protein purification system was more than 90%. Western-blot showed the good antigenicity of the target proteins.
Show more [+] Less [-]Chinese. 用PCR法扩增了牛疱疹病毒Ⅰ型Bartha Nu/67 株gB基因，将其插入原核表达载体pBAD/TOPO中构建重组质粒pBAD-gB。将pBAD-gB质粒转化大肠杆菌TOP10后进行了诱导表达，对表达产物进行了纯化和抗原性检测，并通过改变诱导剂L-Arabinose的浓度和诱导时间对诱导表达条件进行了优化。结果表明，重组质粒pBAD-gB构建成功，gB蛋白获得了高效表达，并以包涵体形式存在，纯化后gB蛋白的纯度达90%以上，gB蛋白抗原性良好，最佳诱导表达条件:L-Arabinose终浓度为0.2 g/L，诱导时间为5 h。
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