摘要
本研究通过应用基因工程技术,构建融合基因pET14b-Mincle的原核表达载体,并将其转化到大肠杆菌BL21(DE3)中诱导表达目的蛋白Mincle,并对其表达产物进行纯化、鉴定、透析复性。采用RT-PCR技术扩增小鼠Mincle的基因片段,将其克隆于载体pMD18-T中,并克隆到带有His-tag的原核表达载体pET14b中;重组质粒经酶切鉴定、序列比对验证正确后转化大肠杆菌BL21(DE3)中经IPTG诱导表达目的蛋白。经1mmol/LIPTG在37℃下诱导5h获得了分子量约为22kDa的重组融合蛋白的优化表达,SDS-PAGE、Western Blot和ELISA证实了重组蛋白的特异性。Mincle以包涵体形式在宿主中表达,利用Ni2+亲和柱进行纯化和生物膜透析复性,纯化和透析后的蛋白经WesternBlot、ELISA法定性鉴别以及用白色念珠菌(SC5314)整个灭活细胞对复性后蛋白的活性测定,透析后的Mincle重组融合蛋白能与白色念珠菌的特异性结合体现其生物活性,表明获得了具有活性的蛋白,为后续研究打下基础。
In this study, genetic engineering techniques were used to construct the recombinant prokaryotic expression vector p ET14b-Mincle, which was then transformed into Escherichia colihost strain BL21（DE3） to induce the expression of the target protein Mincle. The expression product was purified, verified, and refolded using dialysis. Mincle coding sequence fragments were amplified by reverse transcript polymerase chain reaction（RT-PCR）, and cloned into the carrier p MD18-T and the prokaryotic expression vector p ET14b which containing His-tag. The constructed vector, verified by restriction endonuclease digestion and DNA sequence comparisons, was then transformed into E. colihost strain BL21（DE3） and expression was induced using IPTG. A recombinant fusion protein with a molecular weight of approximately 22k Da was expressed under optimized induction conditions with 1mmol/L IPTG at 37 ℃ for 5h. The specificity of the recombinant protein was confirmed using SDS-PAGE, western blots, and ELISA. Mincle was expressed in thehost in the form of inclusion bodies, and was purified using a Ni2 ＋ affinity column and refolded by biofilm dialysis. The purified and dialyzed protein was verified by western blotting and ELISA, and its activity was examined with inactivated whole cells of Candida albicans（SC5314）. The specific binding with C. albicans showed that the Mincle recombinant fusion protein was biologically active. Thus, this method produced active protein and forms a basis for future studies.
出处
《现代食品科技》
EI
CAS
北大核心
2015年第3期77-83,76,共8页
Modern Food Science and Technology
基金
中央财政支持地方高校发展专项资金项目(粤财教[2010]358号)
教育部高等学校博士学科点专项科研基金(20114425120010)
国家自然科学基金项目(31201330)
广州市科技攻关项目(201300000202)
