肺炎支原体GrpE蛋白的生物信息学分析、纯化及多克隆抗体的制备

Bioinformatics analysis and purification of Mycoplasma pneumoniae GrpE protein and preparation of polyclonal antibody

目的利用生物信息学方法分析肺炎支原体GrpE蛋白的生物学特性,纯化MpGrpE蛋白并制备多克隆抗体。方法从NCBI网站获得肺炎支原体GrpE蛋白的氨基酸序列,并将其与大肠埃希菌、结核分枝杆菌、解脲脲原体、生殖支原体、鸡毒支原体和龟支原体的GrpE蛋白进行序列比对。利用生物信息学软件分析预测MpGrpE蛋白的信号肽、跨膜区、细胞定位、磷酸化和糖基化修饰、二级结构、三级结构、B细胞表位和作蛋白等生物学特性。将重组质粒pET28a-MpGrpE转化大肠埃希菌,诱导表达后通过Ni亲和层析纯化重组MpGrpE蛋白。用重组蛋白免疫小鼠,获得抗血清并测定抗体滴度。结果MpGrpE蛋白与生殖支原体GrpE蛋白同源性为73%。MpGrpE蛋白共有217个氨基酸,分子式CHNOS,理论分子质量为24.7ku,理论等电点为7.7,为不稳定的疏水性蛋白。MpGrpE蛋白无信号肽,无跨膜区,定位在胞质。该蛋白含有8个丝氨酸、4个苏氨酸和2个酪氨酸磷酸化位点,无糖基化位点。其二级结构中α螺旋占69.12%,β折叠占8.29%,无规卷曲占18.89%,β转角占3.69%。MpGrpE蛋白83.2%的三级结构为最佳状态。MpGrpE蛋白含有4个连续的B表位和4个非线性表位。MpGrpE蛋白的的互作蛋白有DnaK、GroS、GroL等。通过Ni亲和层析获得了较高纯度的MpGrpE蛋白,用该蛋白免疫小鼠能够诱导产生IgG抗体。结论生物信息学分析MpGrpE蛋白是定位在肺炎支原体胞质的一种疏水性蛋白,克隆表达的MpGrpE蛋白具有良好的免疫原性,为肺炎支原体的致病机制以及疫苗研发提供了实验基础。

Objective To analyze the biological characteristics of Mycoplasma pneumoniae GrpE protein,purify GrpE protein and prepare polyclonal antibody.Methods The amino acid sequence of MpGrpE protein was obtained from NCBI website,which was used to compare with the amino acids of GrpE protein from E.coli,My.tuberculosis,U.urealyticum,M.genitalium,M.gallisepticum and M.tortoise.Bioinformatics software was used to analyze and predict the biological characteristics of MpGrpE protein,such as signal peptide,transmembrane region,cell localization,phosphorylation and glycosylation modification,secondary structure,tertiary structure,B cell epitope and acting protein.The recombinant plasmid pET28 a-MpGrpE was transformed into E.coli,and the recombinant MpGrpE protein was induced and purified by Niaffinity chromatography.The recombinant protein was immunized with mice to obtain antiserum and determine the antibody titer.Results The homology between MpGrpE protein and GrpE protein of M.genitalium was as high as 73%.MpGrpE protein is an unstable hydrophobic protein,which has 217 amino acids in total,with molecular formula CHNOSand theoretical molecular weight is 24.710~3 ku,the theoretical isoelectric point is 7.7.MpGrpE protein has no signal peptide and transmembrane region,which is located in the cytoplasm.It may contain 8 serine,4 threonine,2 tyrosine phosphorylation sites and no glycosylation sites.The content ofα-helix is 69.12%.The content of random coil was 18.89%,β-sheet was 8.29%andβ-turn was 3.69%.The 83.2%of the tertiary structure of MpGrpE protein was in the best state.MpGrpE protein contains four continuous B epitopes and four nonlinear epitopes.The interacting proteins of MpGrpE protein include DnaK,Gros,grol and so on.High purity MpGrpE protein was obtained,which can induce mice to produce IgG antibody.Conclusion MpGrpE protein is a hydrophobic protein localized in the cytoplasm of M.pneumoniae and has good immunogenicity.This paper provides an experimental basis for the pathogenesis of M.pneumoniae and vaccine research and development.