外源丙氨酸提高蜘蛛牵引丝蛋白天然基因在原核系统中的表达

蜘蛛丝蛋白天然基因的体外表达受诸多因素的限制。本研究在获得生长于中国的Nephila clavipes蜘蛛牵引丝蛋白Spidroin2 cDNA（Genbank Accession No. AF441245）的基础上，利用限制性内切酶双酶切反应构建含有Spidroin2 cDNA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Spidroin2 cDNA的表达量，同时利用多克隆抗体对表达产物进行Western blot检测。重组质粒pET-28b(+)-Sp的测序结果表明Spidroin2 cDNA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31 kDa的目的蛋白带（加入外源丙氨酸条件下），Western blot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。本研究证实，蜘蛛牵引丝蛋白Spidroin2 cDNA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。

Exogenous Alanine Enhances the Expression of Spidroin2 cDNA of Spider Dragline Silk Protein in Escherichia coli

Abstract Expression in vitro of the natural genes of spider silk protein is limited by many factors. Based on the cloning of Spidroin2 cDNA of Nephila clavipes (Genbank Accession No. AF441245), we constructed a prokaryotic expression vector pET-28b(+)-Sp by double digestion. The recombinant plasmids pET-28b(+)-Sp were then transferred into the competent expression host strain BL21(DE3) E. coli. Expression of the native gene Spidroin2 cDNA was induced by the addition of different concentrations of IPTG, followed by extension of induction time, culture temperature and addition of exogenous alanine to increase the expression of interest gene. The recombinant protein was identified by SDS-PAGE and Western blot in which the polyclonal antiserum of dragline silk protein was used as the first antibody. Sequencing result of recombinant expression plasmid showed that Spidroin2 cDNA was inserted into the prokaryotic expression vector pET-28b(+) with a correct read frame. An interest protein with a molecular weight of 31 kDa was observed on SDS-PAGE gel in the condition of alanine addition, which can be specifically immunoblotted with a polyclonal antibody targeted at spider silk protein. We concluded that the Spidroin2 cDNA of Nephila clavipes can be expressed in prokaryotic expression system, and addition of exogenous alanine plays important role in the promotion of expression of the naive gene of spider silk protein.