曼氏迭宫绦虫半胱氨酸蛋白酶体外表达条件的优化

The in vitro expression conditions of Spirometra mansoni cysteine protease

目的优化曼氏(欧猥)迭宫绦虫半胱氨酸蛋白酶(Spirometra erinaceieuropaei cysteine protease,SeCP)基因的体外表达条件。方法对含有SeCP基因重组表达质粒pMAL-c2X-SeCP的大肠埃希菌TB1在不同培养温度、不同诱导剂异丙基硫代β-D-半乳糖苷(isopropyl-1-thio-β-galactopyranoside,IPTG)浓度及不同培养时间等条件下进行诱导表达,通过SDS-PAGE与蛋白图谱扫描分析重组SeCP蛋白(rSeCP)的表达水平,选择rSeCP适宜的体外表达条件。结果SDS-PAGE显示重组质粒pMAL-c2X-SeCP转化大肠埃希菌TB1在常规诱导条件(培养温度30℃,1 mmol/L IPTG诱导培养4h)下,rSeCP以可溶性蛋白和包涵体2种形式表达。当重组菌TB1在28、30、31、32、34、35及37℃条件下培养时,rSeCP的表达量分别占全菌上清蛋白量的9.4%、15.1%、12.2%、6.6%、6.4%、5.4%和1.2%;当重组菌TB1在30℃条件下IPTG终浓度0.1、0.2、0.5及1.0mmol/L诱导时,rSeCP表达量分别占全菌总蛋白量的32.6%、25.7%、26.7%和25.7%;当重组菌TB1在30℃条件下IPTG终浓度0.1mmol/L诱导培养1、2、3、4、5和6h,rSeCP的表达量分别占全菌总蛋白量的13.4%、18.6%、22.4%、33.2%、45.2%和34.6%。结论培养温度为30℃,IPTG终浓度为0.1mmol/L诱导培养5h,为重组质粒pMAL-c2X-SeCP转染大肠埃希菌TB1表达rSeCP最佳条件,该研究为大量制备和纯化rSeCP奠定了基础。

Objective To optimize the in vitro expression conditions of Spirometra mansoni cysteine protease（SeCP）. Methods The Escherichia coli TB1 harboring the recombinant expression plasmid pMAL-c2X-SeCP of the SeCP gene was induced under the in vitro different culture temperature,concentration of isopropyl-1-thio-β-galactopyranoside（IPTG）and culture time,the expression levels of the recombinant SeCP protein（rSeCP）were investigated by SDSPAGE and thin-layer gel optical scanning analysis. Results The results of SDS-PAGE analysis showed that the rSeCP was present in two forms of the soluble proteins and inclusion bodies in E.coli TB1 when the recombinant TB1 harboring the recombinant plasmid pMAL-c2X-SeCP was induced under the routine induction conditions（1mmol/L IPTG,inducing at 30 ℃for 5h）.The portion of the rSeCP accounted for 9.4%,15.1%,12.2%,6.6%,6.4%,5.4% and 1.2% of the total bacterial proteins in the supernatant when the recombinant bacterium TB1 were cultured at 28,30,31,32,34,35 and 37 ℃,respectively.However,when the recombinant TB1 were induced at different final concentrations of IPTG（0.1,0.2,0.5and 1.0mmol/L）,the amount of the rSeCP accounted for 32.6%,25.7%,26.7% and 25.7% of total bacterial proteins,respectively.Additionally,while the recombinant TB1 were cultured at 30 ℃ and 0.1mmol/L IPTG for 1,2,3,4,5and 6h,the rSeCP respectively accounted for 13.4%,18.6%,22.4%,33.2%,45.2%and 34.6% of total bacterial proteins. Conclusion The in vitro optimal expression conditions of rSeCP were that the recombinant E.coli TB1TB1 harboring the recombinant plasmid pMAL-c2X-SeCP was induced at 30 ℃ and 0.1mmol/L IPTG for 5h.This study provided the basis for expression and purification of a plenty of the rSeCP in the future.