肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中的表达及应用研究

目的:克隆、表达和鉴定肺炎支原体(Mycoplasma pneumoniae,MP)P1蛋白羧基端基因序列,为制备抗体和基因工程疫苗打下基础。方法 在成功克隆肺炎支原体P1蛋白羧基端基因片段并测序的基础上,将基因序列克隆到表达载体pET32a(+)上,构建了重组表达质粒pET32a(+)/P1(3 520~4 563bp),转化大肠杆rosetta,IPTG诱导表达,利用Ni^2＋亲和层析柱对重组蛋白进行纯化,并用ELISA和Western blotting方法检测其抗原性。重组蛋白免疫小鼠,制备单克隆抗体。结果 重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致,蛋白质纯度达95％以上。ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。并成功获得两株高效价单克隆抗体。结论:本研究成功克隆和表达了肺炎支原体P1蛋白羧基端基因序列,制备了抗肺炎支原体P1蛋白单克隆抗体,为肺炎支原体诊断试剂和疫苗的开发等进一步的研究奠定了基础。

Expression and Application Research of the C-terminal Gene Fragment of P1 Protein of Mycoplasma pneumoniae in E.coli

Objective:To clone,express and characterize the C-terminal gene fragment of P1 protein.Methods:On the basis of successful clone and sequence analysis of the C-terminal gene fragment of P1,the gene fragment were ligated into pET32a(+).An expression vector pET32a(+)/P1(3 520~4 563bp)were constructed and expressed in E.coli rosetta induced by IPTG.recombinant protein was purified through GSTrap 4B affinity chromatography column.ELISA and Western blotting were used to determine the antigenic of the recombinant ...