穿透支原体膜蛋白MYPE2350的表达及免疫活性分析  

Expression and analysis of the immune activity of the membrane protein MYPE2350 of Mycoplasma penetrans

作　　者：侯权芳 谭潭[2] 张健[2] 李芬[2] 罗良贤[2] 李冉辉 HOU Quan-fang;TAN Tan;ZHANG Jian;LI Fen;LUO Liang-xian;LI Ran-hui(Institute of Pathogen Biology,Hengyang Medical School,University of South China,Hengyang,Hunan 421001,China;The First People’s Hospital of Chenzhou,Chenzhou;The Affiliated Chenzhou Hospital,Hengyang Medical,School,University of South China)

机构地区：[1]南华大学衡阳医学院病原生物学研究所,湖南衡阳421001 [2]郴州市第一人民医院 [3]南华大学衡阳医学院附属郴州医院

出　　处：《中国病原生物学杂志》2021年第10期1173-1177,共5页Journal of Pathogen Biology

基　　金：湖南教育厅优秀青年项目(No.19B496);湖南省自然科学基金项目(No.2020JJ4521);湖南省卫健委科学基金项目(No.20200706);衡阳市自然科学基金项目(No.2019-117)。

摘　　要：目的对穿透支原体膜蛋白MYPE2350进行生物学信息学分析,构建重组原核表达质粒,表达、纯化并检测该蛋白的免疫学活性。方法从NCBI网站获得穿透支原体特有MYPE2350蛋白的氨基酸序列,利用生物信息学软件预测和分析MYPE2350蛋白的理化性质、跨膜区、信号肽和B细胞表位等生物学特性。设计引物,PCR扩增MYPE2350基因片段,将其连接到质粒pET28a。将重组质粒pET28a-MYPE2350转化入大肠埃希菌BL21,用IPTG诱导重组MYPE2350蛋白的表达。分离包含体,使用尿素溶解后利用Ni^(2+)亲和层析法纯化重组MYPE2350蛋白。用重组MYPE2350蛋白免疫小鼠,测定小鼠血清特异IgG含量和IgG2a/IgG1的比值,以及小鼠脾细胞分泌细胞因子的含量。结果 MYPE2350蛋白共有178个氨基酸,相对分子质量为19.4×10^(3),为跨膜蛋白,具有3个跨膜区。该蛋白二级结构中α螺旋占43.82%,β折叠占17.42%,无规卷曲占35.39%,β转角占3.37%。Swiss网站预测的蛋白三级结构的匹配度和可信度不高。MYPE2350蛋白含有多个B细胞表位。构建的重组质粒pET28a-MYPE2350转化BL21后,经IPTG诱导表达重组MYPE2350蛋白,通过亲和层析纯化获得了纯度较高的MYPE2350蛋白。重组MYPE2350蛋白具有良好的免疫原性,能够诱导小鼠分泌特异性抗体,且IgG2a/IgG1<1。MYPE2350蛋白能诱导小鼠脾细胞IL-4和IL-5(Th2型细胞因子)高表达,但对IFN-γ和TNF-α(Th1型细胞因子)的影响较小。结论克隆表达的重组穿透支原体特有膜蛋白MYPE2350具有良好的免疫原性,能诱导Th2型免疫应答。Objective To analyzed biological information on the membrane protein MYPE2350 of Mycoplasma penetrans, to construct a recombinant prokaryotic expression plasmid, to express and purify the recombinant MYPE2350 protein, and to test its immunological activity. Methods The amino acid sequence of the MYPE2350 protein of M. penetrans was obtained from the NCBI website. The physical and chemical properties, transmembrane domains, signal peptides, and B-cell epitopes of the MYPE2350 protein were analyzed using bioinformatic software. PCR primers were designed, and the MYPE2350 gene was amplified with PCR. The gene was ligated to the plasmid pET28 a. The recombinant plasmid pET28 a-MYPE2350 was transformed into E. coli BL21, and expression of the recombinant MYPE2350 protein was induced with IPTG. The inclusion bodies were separated and dissolved in urea, and the recombinant MYPE2350 protein was purified using Ni^(2+)affinity chromatography. Mice were immunized with the recombinant MYPE2350 protein. The total IgG content and the ratio of IgG2 a/IgG1 in mouse serum were determined. The levels of cytokines secreted by splenocytes were measured using ELISA. Results The MYPE2350 protein is a protein with three transmembrane domains, 178 amino acids, and a molecular weight of 19.4×10^(3). α-helices account for as much as 43.82% of the protein’s secondary structure, random coils account for 35.39%, β-sheets account for 17.42%, and β-turns account for 3.37%. The tertiary structure predicted using the Swiss website was discordant and not very reliable. The MYPE2350 protein contains multiple B-cell epitopes. The recombinant plasmid pET28 a-MYPE2350 was successfully constructed. Expression of the recombinant MYPE2350 protein was induced with IPTG, and the MYPE2350 protein was purified using affinity chromatography. The recombinant MYPE2350 protein has good immunogenicity and can induce mice to secrete specific antibodies, with a IgG2 a/IgG1 ratio of less than 1. The MYPE2350 protein can induce a high level of expression of IL

关 键 词：穿透支原体 膜蛋白 生物信息学分析 免疫原性 

分 类 号：R375[医药卫生—病原生物学]