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目的:克隆肝素结合血凝素(HBHA)基因,并在大肠杆菌中进行表达和纯化,利用获得的蛋白进行免疫学特性的初步研究。方法:用PCR方法从结核分枝杆菌H37Rv基因组扩增出HBHA基因片段,克隆至pMD18-T载体中,序列测定正确后,将其亚克隆到表达载体pQE80L并在大肠杆菌DH5α中表达,表达蛋白经SDS-PAG及Western-blot分析后,亲和层析法纯化蛋白。获得的蛋白免疫BALB/c小鼠,测定血清抗体水平及IgG2a/IgG1比例。结果:克隆了HBHA基因,并成功表达该蛋白,SDS-PAGE及Western-blot分析表明表达产物正确。通过亲和层析法得到28kD纯化蛋白,与文献报道相符,诱导小鼠可使CD4+和CD8+细胞数明显增加。结论:成功获得了纯化的HBHA蛋白,明确了HBHA蛋白的免疫学特性,为进一步研究HBHA蛋白的致病机理及新型疫苗的开发提供了实验依据。
OBJECTIVE: To clone and clone the hemagglutinin (HBHA) gene and express and purify it in Escherichia coli. The immunogenicity of the obtained protein was studied. Methods: HBHA gene fragment was amplified by PCR from Mycobacterium tuberculosis H37Rv genome and cloned into pMD18-T vector. After sequencing, the HBHA gene fragment was subcloned into expression vector pQE80L and expressed in E. coli DH5α, After SDS-PAGE and Western-blot analysis, the protein was purified by affinity chromatography. BALB / c mice were immunized with the obtained protein, and serum antibody level and IgG2a / IgG1 ratio were measured. Results: HBHA gene was cloned and successfully expressed. SDS-PAGE and Western-blot analysis showed that HBHA gene was expressed correctly. The 28kD purified protein was obtained by affinity chromatography, which accorded with the reported in the literature. The induced CD4 + and CD8 + cells were significantly increased in mice. CONCLUSION: The purified HBHA protein was successfully obtained and the immunological characteristics of HBHA protein were clarified. The results provide experimental evidence for further study on the pathogenesis of HBHA protein and the development of new vaccines.