蜘蛛牵引丝蛋白MaSp1原核表达载体构建及其在大肠杆菌中的表达与纯化

目的通过基因工程手段实现牵引丝关键组成蛋白MaSp1在大肠杆菌中的异源表达,并对其进行分离纯化,从而建立基因工程蜘蛛丝基因序列串联拼接、载体构建以及原核表达与纯化的关键技术体系。方法利用同尾酶连接法对合成的基因工程蜘蛛丝基因单体序列进行串联拼接,获得多倍串联体克隆重组子,将鉴定正确的多倍串联体克隆重组子与原核表达载体pET28a（＋）连接,转化至大肠杆菌BL21（DE3）,IPTG诱导其表达,表达产物通过SDS-PAGE和Western印迹进行鉴定。在此基础上对工程菌进行高密度发酵,所获蛋白通过硫酸铵分级分离方法进行纯化。结果与结论成功构建了基因工程蜘蛛丝MaSp1多串联体的表达载体,原核表达蛋白的相对分子质量与预期一致,且纯化的目的蛋白纯度达80%以上。上述研究工作为开展基因工程蜘蛛丝蛋白的规模化制备建立了关键技术方法,并为后续基因工程蜘蛛丝的人工纺丝提供必要的前提和工作基础。

Construction of spider draggling silk protein MaSp1 prokaryotic expression vector and its expression and purification in Escherichia coli

Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a（＋）, and then transformed into E.coli BL21 （DE3）.After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .