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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 Mannich’s Escherichia coli (RSV) spirometra erinaceieuropaei cysteine protease (SeCP) gene. Methods Escherichia coli TB1 harboring SeCP gene recombinant plasmid pMAL-c2X-SeCP was cultured in different culture temperature and different inducer isopropyl-1-thio-β-galactopyranoside IPTG) and different culture time. The expression level of recombinant SeCP protein (rSeCP) was analyzed by SDS-PAGE and protein profiling. The suitable conditions of rSeCP expression in vitro were selected. Results SDS-PAGE showed that rSeCP was expressed in two forms of soluble protein and inclusion body under the conditions of conventional induction (incubation temperature 30 ℃, 1 mmol / L IPTG induction culture for 4 h), and the recombinant plasmid pMAL-c2X-SeCP transformed Escherichia coli TB1 . When the recombinant bacteria TB1 was cultured at 28, 30, 31, 32, 34, 35 and 37 ℃, the expression of rSeCP accounted for 9.4%, 15.1%, 12.2%, 6.6% and 6.4% %, 5.4% and 1.2%, respectively. When the recombinant strain TB1 was induced by 0.1,0.2,0.5 and 1.0 mmol / L IPTG at 30 ℃, the expression of rSeCP accounted for 32.6% and 25.7% 26.7% and 25.7% respectively. When the recombinant strain TB1 induced the culture for 1, 2, 3, 4, 5 and 6 h with 0.1 mmol / L IPTG at 30 ℃, the expression of rSeCP accounted for 13.4% , 18.6%, 22.4%, 33.2%, 45.2% and 34.6% respectively. CONCLUSION: The optimum conditions for the expression of rSeCP in Escherichia coli TB1 transfected with recombinant plasmid pMAL-c2X-SeCP were established under the conditions of 30 ℃ culture and 0.1 mmol / L IPTG final concentration induction. This study lays the foundation for the preparation and purification of rSeCP basis.