曼氏迭宫绦虫果糖-1,6-二磷酸酶的生物信息学分析、基因克隆及蛋白表达  

Bioinformatic analysis, gene cloning, and protein expression of fructose-1,6-bisphosphatase from Spirometra mansoni

作　　者：王清吟 谭钦月 符瑞佳[1,2] 周晓君[5] 刘亚妹[1] 王妹妹 林于金 左世亮 韩媛 尹飞飞 吕刚[1,2] 梁培[1,2] WANG Qing-yin;TAN Qin-yue;FU Rui-jia;ZHOU Xiao-jun;LIU Ya-mei;WANG Mei-mei;LIN Yu-jin;ZUO Shi-liang;HAN Yuan;YIN Fei-fei;LV Gang;LIANG Pei(General Hospital,Association of People’s Hospitals in the Fenghua District,Zhejiang,Ningbo 315500,China;Guangyuan Central Hospital;School of Tropical and Laboratory Medicine,Hainan Medical University;Key Laboratory of Translational Medicine for Tropical Diseases,Ministry of Education,Hainan Medical University;Hainan General Hospital)

机构地区：[1]海南医学院热带医学与检验医学院,海南海口571199 [2]海南医学院热带病转化医学教育部重点实验室 [3]宁波市奉化区人民医院共体总院,浙江宁波315500 [4]广元市中心医院 [5]海南省人民医院

出　　处：《中国病原生物学杂志》2020年第7期805-811,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81560332);海南省科协青年科技英才学术创新计划项目(No.QCXM201918);海南省高校科学研究项目(No.SJK180005);海南省大学生创新创业训练计划项目(No.201811810064);海南医学院大学生创新创业训练计划项目(No.HYCX2018066&No.HYCX2018151)。

摘　　要：目的通过生物信息学分析预测曼氏迭宫绦虫果糖-1,6-二磷酸酶(SmFBPase)的生物学特性,同时对SmFBPase进行基因克隆及蛋白原核表达。方法通过NCBI网站对SmFBPase基因序列进行开放阅读框分析和核苷酸序列同源性分析。通过ExPASy网站预测SmFBPase的理化性质、结构及表位等特性。构建pET-28α-SmFBPase重组质粒,并转化入~E.coli^BL21(DE3)菌株,用异丙基-β-D-硫代半乳糖苷对其诱导表达,用Western blot进行鉴定。结果SmFBPase由337个氨基酸组成,无信号肽、无跨膜区,理论相对分子质量约37×10~3,为稳定胞内蛋白。SmFBPase的氨基酸序列与人FBPase的氨基酸序列一致性为61.77%,且进化树中两者亲缘性较远,该蛋白含有13个B细胞表位。PCR、双酶切鉴定及测序分析,证明重组质粒构建成功。Western blot显示重组质粒转化BL21后成功诱导表达SmFBPase蛋白。结论生物信息学分析SmFBPase是一个具有潜在研究价值的药物靶点,原核表达的SmFBPase具有反应原性,为研究其糖代谢规律奠定了基础。Objective To predict biological characteristics of fructose-1,6-bisphosphatase from Spirometra mansoni(SmFBPase)using bioinformatic analysis.And to clone SmFBPase into a prokaryotic system and express the recombinant protein.Methods Open reading frame analysis(ORF)and analysis of the nucleotide sequence homology of SmFBPase were performed using the NCBI website.ExPASy was used to predict the physical and chemical properties,secondary structure,and signal peptides of the protein.A prokaryotic expression system for expression of the recombinant expression plasmid pET-28α-SmFBPase was successfully constructed.Expression of the recombinant protein was induced with 1 mmol/L isopropyl-β-D-thiogalactoside(IPTG),and the protein was identified with anti-His-tag mouse serum in Western blotting.Results SmFBPase consisted of 337 amino acids,no signal peptides,and no transmembrane regions.SmFBPase is a stable intracellular protein with a theoretical molecular mass of approximately 37×10~3.The amino acid sequence similarity between SmFBPase and FBPases from humans was 61.77%,and the two are distant relatives in the evolutionary tree.The amino acid sequence of SmFBPase contained 13 B-cell epitopes.The recombinant plasmid was confirmed as correct using PCR,double enzyme digestion,and DNA sequencing.Western blotting indicated that the recombinant protein was successfully expressed.Conclusion Bioinformatic analysis indicated that SmFBPase is a potential drug target.In addition,the protein was successfully expressed,laying a material foundation for the study of the glucose metabolism of parasites in a further study.

关 键 词：曼氏迭宫绦虫 果糖-1 6-二磷酸酶 生物信息学分析 蛋白原核表达 药物靶点 

分 类 号：R512.91[医药卫生—内科学]