摘要
蜘蛛丝蛋白天然基因的体外表达受诸多因素的限制。在获得生长于中国的Nephilaclavipes蜘蛛牵引丝蛋白Spidroin2cDNA(No.AF441245)的基础上,利用限制性内切酶双酶切反应构建含有Spidroin2cDNA的重组表达质粒pET-28b(+)-Sp。将该质粒转化至大肠杆菌BL21(DE3)宿主细胞感受态菌中,以不同浓度的IPTG进行诱导,并通过诱导时间、培养温度、加入外源丙氨酸等途径提高Spidroin2cDNA的表达量,同时利用多克隆抗体对表达产物进行Westernblot检测。重组质粒pET-28b(+)-Sp的测序结果表明Spidroin2cDNA基因以正确的阅读框插入到原核表达载体中;SDS-PAGE结果表明菌体表达蛋白中存在着大小约为31kDa的目的蛋白带(加入外源丙氨酸条件下),Westernblot检测结果进一步证实,目的基因在大肠杆菌中得到正确表达。实验证明,蜘蛛牵引丝蛋白Spidroin2cDNA可在原核细胞内正确表达,外源丙氨酸的加入对于提高天然蜘蛛丝蛋白基因在原核系统的表达作用明显。
Spider dragline silk, possesses unmatched high mechanical strength and elasticity which make it famous as ＂ Bio-steel＂ or ＂ Bio-rubber＂, is a very promising biomaterial. Native genes encoding the spider dragline silk proteins were proved to be low expressions in vitro limited by many factors. A recombinant prokaryotic expression vector containing Spidroin2 cDNA of Nephila clavipes[ pET-28b（ ＋ ）-Sp] was constructed, and then transferred into the competent expression host strain BL21 （DE3）E. coll. Expression of the native gene Spidroin2 cDNA was induced by adding different concentrations of IPTG and exogenous alanine, at different culture temperature for different induction time to get an optimal expression condition. The results showed that higher expression of the interest protein with a molecular weight of 32kDa was observed on SDS-PAGE when alanine existed in the culture medium, which can be specifically immunoblotted with a polyclonal antibody targeted at spider silk protein, while no increase of expression yield was observed in other conditions. It was concluded that the Spidroin2 cDNA of Nephila clavipes could be expressed in prokaryotic expression system, and addition of exogenous alanine played a very important role in the promotion of expression of the native gene of spider silk protein.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第1期47-51,共5页
China Biotechnology
关键词
蜘蛛
牵引丝蛋白
天然基因
原核表达
Spider Dragline silk protein Native gene Prokaryotic expression
