重组蛋白EgG1Y162-GGGGSGGG-EgG1Y162的原核表达、纯化及鉴定

Construction of the pET30a-EgG1Y162-GGGGSGGG-EgG1Y162 and identification of the purified expression products

目的利用pET30a-EgG1Y162-GGGGSGGG-EgG1Y162重组质粒,诱导表达、纯化获得EgG1Y162-GGGGSGGG-EgG1Y162目的蛋白,并对其进行鉴定,为后续研究提供前提条件。方法利用双酶切的方法验证pET30a-EgG1Y162-GGGGSGGG-EgG1Y162质粒,并将其转化至E.coli BL21(DE3)菌株,获得表达菌株。采用不同浓度的IPTG在不同诱导时间及温度下诱导表达重组蛋白,菌液超声破碎后,经SDS-PAGE检测上清及沉淀中蛋白的表达。通过镍柱亲和层析法纯化蛋白并进行Western blot鉴定。结果成功鉴定重组质粒pET30a-EgG1Y162-GGGGSGGG-EgG1Y162;重组蛋白EgG1Y162-GGGGSGGG-EgG1Y162在IPTG终浓度为0.5 mmol/L,28℃条件下诱导6 h,上清中的蛋白表达量最高;300 mmol/L咪唑时洗脱得到的目的蛋白较多,洗脱效果较好;Western blot检测重组蛋白EgG1Y162-GGGGSGGG-EgG1Y162能被相应抗体识别,反应条带在35 ku处,与预期相符。结论成功构建pET30a-EgG1Y162-GGGGSGGG-EgG1Y162重组质粒,表达出相应重组蛋白,为细粒棘球病重组疫苗的研究创造条件。

Objective The recombinant plasmid pET30a-EgG1Y162-GGGGSGGG-EgG1Y162 was induced to express the target protein EgG1Y162-GGGGSGGG-EgG1Y162,and its purification and identification was conducted to provide the prerequisites for the subsequent studies.Methods The pET30a-EgG1Y162-GGGGSGGG-EgG1Y162was validated by restriction enzyme and transformed into E.coli BL21(DE3)strain to obtain the expression strain.The recombinant proteins were induced by IPTG with different concentrations at different induction times and temperature.The bacterial solution was crushed by ultrasound,and the expressed protein in supernatant and precipitation was analyzed by SDSPAGE.The protein was purified by nickel column affinity chromatography and identified by Western blot.Results Theplasmids pET30a-EgG1Y162-GGGGSGGG-EgG1Y162 was successfully identified.The recombinant protein EgG1Y162-GGGGSGGG-EgG1Y162 was induced by IPTG(0.5 mmol/L)at 28℃for 6 hours,and the protein was expressed highly in the supernatant.When it was eluted with 300 mmol/L imidazole,more target proteins are obtained and collected.It was showed by Western blot results that the recombinant protein EgG1Y162-GGGGSGGG-EgG1Y162 could be recognized by the corresponding antibody,and the reaction band was located at 35 ku,which was consistent with the expectation results.Conclusion The pET30a-EgG1Y162-GGGGSGGG-EgG1Y162 was successfullyconstructed,the corresponding recombinant protein wassuccessfully expressed,which established the conditions for the study on the recombinant vaccine for the echinococcosis.