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  

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

作　　者：黄劲松[1] 黄捷勤 唐启慧[2] 陈禹保[2] 

机构地区：[1]北京市科学技术研究院科技处,北京100094 [2]北京标凯科技有限公司,北京100094

出　　处：《中国生物工程杂志》2010年第11期65-69,共5页China Biotechnology

摘　　要：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（3520~4563bp）,转化大肠杆rosetta,IPTG诱导表达,利用Ni2＋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。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 protein.Immune mouse with recombination protein,monoclonal antibody was prepared.Results：The recombinant gene can be overexpressed in E.coli.SDS analysis revealed that the expressed protein was identical to C-terminal gene fragment of P1 protein in molecular weight with a purity of 95%.ELISA and Western blotting analysis showed that the recombinant protein can displayed high antigenic activity toward Mycoplasma pneumoniae convalescent antiserum.Two monoclonal antibody cell line were obtained.Conclusion：The C-terminal gene fragment of P1 protein of MP has been successful cloned and expressed,monoclonal antibody was prepared which could be useful for developing diagnose reagents or vaccine of MP.

关 键 词：肺炎支原体 P1蛋白 克隆表达 单克隆抗体 

分 类 号：Q78[生物学—分子生物学]