细粒棘球蚴重组蛋白EgG1Y162诱导表达条件的 优化及其蛋白结构预测  

Optimization of expression conditions and protein structure prediction of recombinant EgG1Y162 of Echinococcus granulosus

作　　者：陈娜菲 李小坡 马西智 彭学臻 曹春宝 周晓涛 CHEN Nafei;LI Xiaopo;MA Xizhi;PENG Xuezhen;CAO Chunbao;ZHOU Xiaotao(Department of Immunology,School of Basic Medical Sciences,Xinjiang Medical University;Xinjiang Key Laboratory of Endemic Diseases and Molecular Biology,Xinjiang Medical University,Urumqi 830017,China)

机构地区：[1]新疆医科大学基础医学院免疫学教研室 [2]新疆地方病分子生物学重点实验室,乌鲁木齐830017

出　　处：《新疆医科大学学报》2025年第4期426-431,共6页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区杰出青年基金项目(2022D01E50);国家自然科学基金项目(32260192)。

摘　　要：目的探索优化重组质粒pET30a-EgG1Y162表达条件并预测其蛋白结构模型。方法通过使用EgG1Y162原始菌种进行诱导表达,探索不同浓度异丙基硫代半乳糖苷(IPTG,sopropylβ-D-Thiogalactoside)、孵育时间对EgG1Y162蛋白诱导表达水平的影响。原始菌种经诱导表达后,通过超声处理离心得到细菌裂解液上清与沉淀,利用SDS-PAGE分析上清和沉淀中EgG1Y162重组蛋白的表达情况,分析最佳诱导表达条件;纯化蛋白,且通过Western blot鉴定抗原蛋白的表达;利用生物信息学技术预测EgG1Y162蛋白的空间结构模型。结果EgG1Y162蛋白在28℃,IPTG终浓度为0.2 mmoL/L,诱导6 h条件下的表达量较高,蛋白相对分子质量约为25 kDa;用HisTrap纯化柱纯化蛋白,EgG1Y162蛋白在20 mmol/L咪唑浓度的洗脱下,洗脱效果较佳,可获取大量纯化蛋白;纯化蛋白能被鼠抗His-Tag抗体识别,与预期结果相符;生物信息学预测结果显示该蛋白中β折叠占5.00%,无规则卷曲占50.83%,成功构建出EgG1Y162蛋白三级结构模型。结论通过统计学方法获得了重组蛋白EgG1Y162的最佳诱导表达及纯化条件,预测了该蛋白的空间结构,为后续大量获得细粒棘球蚴疫苗抗原蛋白EgG1Y162,进而用于研究其免疫效果与免疫机制创造了条件。Objective To explore and to optimize the expression conditions of recombinant plasmid pET30a-EgG1Y162 and predict the structural model of the protein.Methods The effects of different concentrations of sopropylβ-D-Thiogalactoside(IPTG),incubation time on the induced expression level of EgG1Y162 protein were further explored by using the original EgG1Y162 strain for induced expression.After the original strain was induced to express,the supernatant and precipitate of bacterial lysate were obtained by centrifugation through ultrasonication,and the expression of EgG1Y162 recombinant protein in the supernatant and precipitate were analyzed by SDS-PAGE to analyze the optimal conditions for induced expression.The protein was purified and the expression of the antigenic protein was identified by Western-blot.The spatial structure model of EgG1Y162 protein was predicted using bioinformatics techniques.Results The expression of EgG1Y162 protein was higher at 28℃.The final concentration of IPTG was 0.2 mmoL/L,and the expression level was higher after 6 h induction.The relative molecular mass of the protein was about 25 kDa.The protein was purified with HisTrap purification column.The elution effect of EgG1Y162 protein was better at 20 mmol/L imidazole concentration,and a large amount of purified protein could be obtained.The purified protein could be recognized by murine anti-His-Tag antibody,which was consistent with the expected result.The results of bioinformatics prediction showed thatβ-folding accounted for 5.00%and random curling accounted for 50.83%.The tertiary structure model of EgG1Y162 protein was successfully constructed.Conclusion The optimal expression and purification conditions of recombinant protein EgG1Y162 were obtained by statistical methods,and the spatial structure of the protein was predicted,which created conditions for obtaining a large amount of Echinococcus granulosus vaccine antigen protein EgG1Y162 and further studying its immune effect and immune mechanism.

关 键 词：细粒棘球蚴 EgG1Y162 原核表达条件优化 生物信息学 

分 类 号：R383.3[医药卫生—医学寄生虫学]