细粒棘球蚴重组蛋白HIS-CTLA-4IgV-EgG1Y162-2诱导表达条件探索  

Exploration on the expression conditions of the recombinant protein HIS-CTLA-4IgV-EgG1Y162-2 of echinococcus granulosus

作　　者：孔慧芳 赵商岐 刘斌[2] 周彦霞 李玉娇[2] 曹春宝[1] 丁剑冰[1] 周晓涛[1] KONG Hui-fang;ZHAO Shang-qi;LIU Bing;ZHOU Yan-xia;LI Yu-jiao;CAO Chun-bao;DING Jian-bing;ZHOU Xiao-tao(Basic Medical Sciences School of Xinjiang Medical University,Urumqi 830011,Xinjiang Province,China;The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,Xinjiang Province,China)

机构地区：[1]新疆医科大学基础医学院,乌鲁木齐830011 [2]新疆医科大学第一附属医院,乌鲁木齐830054

出　　处：《寄生虫病与感染性疾病》2022年第1期36-41,共6页Parasitoses and Infectious Diseases

基　　金：国家自然科学基金(项目编号:81760656);新疆维吾尔自治区自然科学基金(项目编号:2018D01C157)。

摘　　要：目的探索细粒棘球蚴重组蛋白HIS-CTLA-4IgV-EgG1Y162-2的最佳诱导表达条件,并对其蛋白结构进行预测。方法构建p ET30a-CTLA-4IgV-EgG1Y162-2重组质粒,将其转化至Ecoli.BL21(DE3)细菌中,选择不同的时间段和温度,用不同浓度的IPTG进行诱导蛋白表达。将得到的菌液通过超声破碎,经离心后用SDS-PAGE分别分析上清和沉淀中重组蛋白HIS-CTLA-4IgV-EgG1Y162-2的表达情况。Western Blot鉴定重组蛋白表达。鉴定成功后进行大量诱导表达。采用生物信息学的方法,使用在线软件SWISS MODEL对HIS-CTLA-4IgV-EgG1Y162-2蛋白三维结构进行预测。结果HIS-CTLA-4IgV-EgG1Y162-2重组蛋白在28℃条件下,诱导4 h,IPTG的浓度为0.2 mmol/L时可溶性蛋白表达量达到最大。蛋白的相对分子量约为29 kD,结果与预期相符。其蛋白三维结构的预测与2×44.1.A结构的相似程度为78.13%。结论探索出HIS-4IgV-EgG1Y162-2重组蛋白最佳诱导表达条件,并成功预测出其蛋白的三维结构模型,为细粒棘球蚴重组疫苗的进一步研究创造条件。objective To explore the best conditions for induction of expression of recombinant proteins echinococcus granulosus HIS-CTLA-4IgV-EgG1Y162-2 and its predicted protein structure.Methods The recombinant plasmid pET30a-CTLA-4IgV-EgG1Y162-2 was constructed and transformed into Ecoli.BL21(DE3).The plasmid was induced to express protein at different time periods and temperatures,induced protein expression was used with different concentrations of IPTG.The obtained bacterial solution was broken up by ultrasound.After being centrifuged and centrifuged,the expression of recombinant protein HIS-CTLA-4IgV-EgG1Y162-2 in supernatant and precipitation was analyzed by SDS-PAGE.The recombinant protein HIS-CTLA-4IgV-EgG1Y162-2 was identified by Western blot.After successful identification,a large number of induced expressions were conducted.The three-dimensional structure of the protein HIS-CTLA-4IgV-EgG1Y162-2 was predicted through the SWISS MODEL on line.Results When the concentration of IPTG was 0.2 mmol/L,the soluble protein expression of the recombinant protein HIS-CTLA-4IgV-EgG1Y162-2 was the highest when it was induced at 28℃for 4 h.The relative molecular weight of the protein was about 29 kD,the results were in line with expectations.The similarity was 78.13%between the recombinant protein and 2×44.1.A in 3D structure of protein.Conclusions The induction condition of the recombinant protein HIS-CTLA-4IgV-EgG1Y162-2 of echinococcosis granulosa was explored,and the 3D structure model of the protein was successfully predicted.It will provide the conditions for further study of recombinant echinococcus granulosus vaccine.

关 键 词：细粒棘球蚴 HIS-CTLA-4IgV-EgG1Y162-2 原核表达 生物信息学 

分 类 号：R383.33[医药卫生—医学寄生虫学]