蝎毒活性肽BmK AS的基因克隆与表达

Gene clone and expression of scorpion active peptide BmK AS

目的克隆东亚钳蝎活性肽BmKAS的基因,在大肠杆菌中进行表达。方法利用PCR技术从蝎尾cDNA pool中扩增BmKAS基因,通过基因工程方法构建表达载体pET28a-BmKAS,在E.coli中进行表达,表达产物为包含体。采用分步稀释复性法对表达产物进行体外变复性。结果BmKAS以包含体的形式得到表达,表达量的质量约占菌体总蛋白质量的31%。表达产物复性后,通过离子交换柱层析进一步纯化,达到了电泳纯,收率为1.6 mg.L-1。结论首次对具有多种药理活性的蝎毒活性肽BmKAS进行了表达,并对表达产物进行体外变复性研究,获得了毫克级的收率,满足了深入研究需要。

Objective To clone the gene of BmK AS and express it in E. coli. Methods The gene of BmK AS was cloned from the cDNA pool by PCR technology. It was reconstructed into the vector pET28a and expressed in E. coll. BL21（DE3）in pellets. The recombinant BrnK AS was renatured in vitro by gradient diluted. The ion exchange chromatography was used to purify the renatured recombinant BrnK AS. Results The gene was cloned and expressed by vector pET28a- BmK AS in EL21 （DE3）. After being denatured and renatured, the recombinant BmK AS was further purified by ion exchange chromatography, and 1.6mg·L^-1 BmK AS was obtained with a purity of 85.5%. Conclusions The scorpion active peptide BmK AS is expressed firstly. An easy approach to get enough BmK AS is provided here. The problem that rare sample of scorpion neurotoxins available for further research is solved.