棘球蚴EM10蛋白T/B联合表位预测分析及表达产物鉴定  

Prediction and analysis of T/B combined epitope of EM10 protein in Echinococcus multilocularis and identification of expressed products

作　　者：马西智 李艳敏 陈娜菲 祖力胡马·艾麦提 王嘉祯 周晓涛 Ma Xizhi;Li Yanmin;Chen Nafei;Zulihuma Aimaiti;Wang Jiazhen;Zhou Xiaotao(Department of Immunology,Basic Medical College,Xinjiang Medical University,Urumqi 830011,China;The First Clinical Medical College,Xinjiang Medical University,Urumqi 830011,China;Key Laboratory of Molecular Biology of Xinjiang Endemic Diseases,Urumqi 830011,China)

机构地区：[1]新疆医科大学基础医学院免疫学教研室,乌鲁木齐830011 [2]新疆医科大学第一临床医学院,乌鲁木齐830011 [3]新疆地方病分子生物学重点实验室,乌鲁木齐830011

出　　处：《中华地方病学杂志》2024年第10期796-802,共7页Chinese Journal of Endemiology

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

摘　　要：目的预测分析棘球蚴EM10蛋白的T/B联合表位,并鉴定生物合成EM10多表位重组蛋白表达产物。方法通过NCBI GenBank公共数据库获得EM10蛋白的基因相关信息。采用生物信息技术预测分析EM10蛋白的T/B联合表位,根据预测结果,生物合成原核表达重组质粒pET30a-EM10(epitope),转化至大肠埃希菌BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导表达,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法鉴定EM10多表位重组蛋白表达产物。结果EM10基因全长1759 bp(GenBank登记号:U05573),其蛋白氨基酸序列(GenBank登记号:AAA50580.1)全长559个氨基酸。Phyre软件同源建模,获得EM10蛋白三级结构,预测EM10蛋白优势T/B联合表位位于46~61、133~183、239~255、442~475位氨基酸。利用(GGGGS)n linker序列连接各表位,设计构建EM10多表位重组蛋白,共206个氨基酸,DNA片段长度为618 bp,蛋白相对分子质量为22.66×10^(3)。构建的原核表达重组质粒pET30a-EM10(epitope),经双酶切鉴定,质粒大小为5000~6000 bp,与预期(5854 bp)相符。SDS-PAGE显示,在37℃IPTG诱导条件下上清中有目的蛋白表达且效果较好;蛋白质印迹法鉴定,表达产物相对分子质量为22.66×10^(3),与预期相符。结论利用生物信息技术成功预测并设计了EM10蛋白的优势T/B联合表位,构建的原核表达重组质粒转化诱导表达产物经实验证实为EM10多表位重组蛋白,为构建EM10优势表位诊断试剂盒提供了实验基础。Objective To predict and analyze the T/B combined epitope of EM10 protein in Echinococcus multilocularis,and identify the expressed products of the biosynthetic EM10 multi epitopes.Methods The gene-related information of EM10 protein was obtained through NCBI GenBank public database.Bioinformatics technique was used to predict and analyze the T/B binding epitopes of EM10 protein.The prokaryotic expession recombinant plasmid pET30a-EM10(epitope)was synthesized,and transformed into host bacteria Ecoli.BL21(DE3).The expression of EM10 recombinant multi-epitope protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting after induced expression by isopropyl thiogalactopyranoside(IPTG).Results The total length of EM10 gene was 1759 bp(GenBank registration number:U05573),and its protein amino acid sequence(GenBank registration number:AAA50580.1)was 559 amino acids.By using Phyre software for homology modeling,the tertiary structure of EM10 protein was obtained,and the T/B combined epitope of EM10 protein was successfully predicted,the dominant epitope was located at 46-61,133-183,239-255 and 442-475 amino acid sites.The(GGGGS)n linker sequence was used to connect the epitopes to form an EM10 recombinant multi-epitope protein with a total of 206 amino acid.The size of the DNA fragment was 618 bp and the relative molecular weight of the protein was 22.66×10^(3).The prokaryotic expession recombinant plasmid was validated by enzyme digestion,the results showed that the plasmid size was between 5000 and 6000 bp,which was consistent with the length of the constructed plasmid(5854 bp).SDS-PAGE showed that the target protein was expressed in the supernatant induced by IPTG at 37℃and the effect was the best.The relative molecular weight of the protein was 22.66×10^(3) by Western blotting,which was consistent with the constructed plasmid.Conclusions The combined epitope of EM10 T/B is successfully designed and predicted using bioinformatics technology.A prokaryotic expres

关 键 词：棘球蚴病 免疫诊断 重组质粒 

分 类 号：R383.2[医药卫生—医学寄生虫学] R383.3[医药卫生—基础医学]