三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

Effects of three promoting labels on the soluble expression of giant panda rotavirus structural protein VP6-VP7

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的 VP 6、 VP 7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。

Giant panda rotavirus (GPRV) is the main cause of diarrhea in young giant pandas, and it has a great harm to captive giant pandas. The rotavirus structural protein VP6 is a carrier protein that mediates mucosal immune responses, and VP7 is the major neutralizing antigen in rotavirus structural proteins. Therefore, the fusion expression of VP6-VP7 as a candidate antigen has important significance for the prevention and treatment of this disease. However, the prokaryotic expression of traditional E. coli has the disadvantages of low expression, poor solubility and low purity. In this study, three kinds of fusion tags, aldolase (EDA), glutathione S-transferase (GST) and maltose-binding protein (MBP), were used to obtain GPRV-VP6-VP7 protein with high expression and high purity. The VP 6 and VP 7 genes were constructed by homologous recombination into the expression vector pET21b containing three kinds of facilitating tags, and the recombinant plasmid was transformed into E. coli Rosetta (DE3) competent cells for low temperature induction. The target protein was purified by Ni-column affinity chromatography, and the protein expression was analyzed by SDS-PAGE and Image J. Western blot analysis showed that the recombinant protein was correct and had activity of protein. The experimental results showed that the EDA tag could significantly promote the prokaryotic soluble expression of VP6-VP7 protein and increase the expression of VP6-VP7 protein.