结核分枝杆菌TB10.4与呼吸道合胞病毒F1蛋白的融合表达

目的在大肠杆菌中融合表达结核分枝杆菌(Mycobacterium tuberculosis,MTB)TB10.4蛋白与呼吸道合胞病毒(Respiratory syncytial virus,RSV)F1蛋白。方法从MTB标准菌株H37Rv全基因组中扩增TB10.4(N/X)和TB10.4(N/B)基因,分别插入原核表达载体pET-28a和pET-30a中,构建重组表达质粒TB10.4(N/X)/pET-28a和TB10.4(N/B)/pET-30a;从F/18T/DH5α菌株中扩增F1(B/X)基因,与TB10.4(N/B)/pET-30a质粒连接,构建重组表达质粒TB10.4-F1/pET-30a;将质粒TB10.4(N/X)/pET-28a和TB10.4-F1/pET-30a分别转化感受态E.coliBL21(DE3),IPTG诱导表达;表达的重组蛋白进行Western blot鉴定。结果重组表达质粒TB10.4(N/X)/pET-28a和TB10.4-F1/pET-30a经双酶切及测序证明构建正确;表达的重组TB10.4和TB10.4-F1蛋白相对分子质量约为13 000和59 000,可与鼠抗His单抗特异性结合。结论成功在大肠杆菌中表达了重组融合蛋白TB10.4-F1,为进一步研究其免疫原性及保护性奠定了基础。

Fusion expression of Mycobacterium tuberculosis TB10.4 protein and respiratory syntytial virus F1 protein

Objective The express the TB10.4 protein of Mycobacterium tuberculosis(MTB) fused with F1 protein of respiratory syncytial virus(RSV) in E.coli.Methods TB10.4(N / X) and TB10.4(N / B) genes were amplified from standard strain H37Rv of MTB and inserted into prokaryotic expression vectors pET-28a and pET-30a to construct recombinant plasmids TB10.4(N / X) / pET-28a and TB10.4(N / B) / pET-30a respectively.F1(B / X) gene was amplified from E.coli F / 18T / DH5α strain and inserted into TB10.4(N / B) / pET-30a to construct recombinant plasmid TB10.4-F1 / pET-30a.Recombinant plasmids TB10.4(N / X) / pET-28a and TB10.4-F1 / pET-30a were transformed to competent E.coli BL21(DE3) respectively for expression under induction of IPTG.The expressed recombinant protein was identified by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmids TB10.4(N / X) / pET-28a and TB10.4-F1 / pET-30a were constructed correctly.The expressed recombinant TB10.4 and TB10.4-F1 proteins,with relative molecular masses of about 13 000 and 59 000 respectively,showed specific binding to mouse anti-His monoclonal antibody.Conclusion Recombinant fusion protein TB10.4-F1 was successfully expressed in E.coli,which laid a foundation of further study on its immunogenicity and protection.