肺炎支原体热休克蛋白HSP70的表达、纯化及其免疫原性分析

The prokaryotic expression and immunogenicity of the HSP70 protein from Mycoplasma pneumoniae

目的构建肺炎支原体(MP)热休克蛋白HSP70重组原核表达质粒,纯化并鉴定其免疫原性。方法以肺炎支原体ATCC 29342为模板,利用搭桥PCR扩增HSP70全序列,胶回收纯化后测序验证。使用BamH1和Xho1双酶切PCR产物,构建重组表达质粒pGEX-4T-1-HSP70,转入大肠埃希菌BL21中,经IPTG诱导目的蛋白表达,对表达产物进行12.5%SDS-PAGE和质谱鉴定。利用AKTA explore 100蛋白纯化仪纯化重组HSP70,以此为抗原与MP感染者血清进行Western blot,检测其免疫反应性。结果搭桥PCR成功完成HSP70基因A1728G突变,重组质粒pGEX-4T-1-HSP70构建正确,转化BL21后经IPTG诱导表达分子质量单位92 ku含GST标签的重组HSP70,纯化蛋白经Western blot检测能与感染Ⅰ型和Ⅱ型MP菌株患儿血清发生特异性反应。结论获得重组HSP70完整蛋白,纯化产物具有免疫反应性,为制备高效防治肺炎支原体感染的基因工程疫苗及研究HSP70蛋白的免疫学功能奠定了基础。

Objective To construct a recombinant prokaryotic expression plasmid of heat shock protein HSP70 of Mycoplasma pneumoniae in order to ascertain the antigenicity of HSP70. Methods The full-length sequence of HSP70 was amplified with M. pneumoniae ATCC 29342 as a template. The PCR product was verified using Sanger sequencing The PCR product was digested with restriction enzymes BamH1 and Xho1 to construct the recombinant expression plasmid pGEX-4 T-1-HSP70, which was transfected into Escherichia coli BL21. The recombinant protein was expressed and identified using SDS-PAGE and mass spectrometry. After the recombinant protein HSP70 was purified using AKTA explore 100, it was subjected to Western blotting with the serum of patients infected with M. pneumoniae to determine its antigenicity. Results The HSP70 gene was successfully incorporated into the plasmid pGEX-4 T-1-HSP70, which expressed recombinant HSP70 protein with a molecular weight of 92 ku and a GST tag. Western blotting of the purified HSP 70 protein revealed specific bands with the serum of children infected with genotype Ⅰ and Ⅱ M. pneumoniae isolates. Conclusion In this study, a complete recombinant HSP70 protein was obtained, and the immunogenicity of the HSP70 protein was preliminarily analyzed. These findings have laid the foundation for the future preparation of genetically engineered vaccines to effectively prevent and treat M. pneumoniae and the study of the immunological functions of the HSP70 protein.