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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 identify the carboxyl terminal gene of P1 protein of Mycoplasma pneumoniae (MP), and lay a foundation for the preparation of antibody and genetically engineered vaccine. Methods Based on the successful cloning and sequencing of the carboxyl terminal gene of P1 protein of Mycoplasma pneumoniae, the gene sequence was cloned into the expression vector pET32a (+). The recombinant plasmid pET32a (+) / P1 (3520 ~ 4563bp) was constructed and transformed into the large intestine The recombinant protein was purified by Ni2 + affinity chromatography and the antigenicity was detected by ELISA and Western blotting. Recombinant proteins were used to immunize mice to prepare monoclonal antibodies. Results The recombinant protein was highly expressed in E. coli. SDS-PAGE showed that the relative molecular mass was consistent with the expected size and the protein purity was over 95%. ELISA and Western blotting experiments confirmed that the recombinant protein has good antigenicity. And successfully obtained two high titer monoclonal antibodies. Conclusion: In this study, we successfully cloned and expressed the carboxyl terminal gene of Mycoplasma pneumoniae P1 protein and prepared the monoclonal antibody against P1 protein of Mycoplasma pneumoniae, which lays the foundation for further research on the diagnosis of Mycoplasma pneumoniae and the development of vaccine.