结核分枝杆菌HSP65与IL-2融合蛋白的表达和纯化

Expression and purification of the fusion protein comprising Mycobacterium tuberculosis heat shock protein 65 and human interleukin 2

目的获得融合表达的结核分枝杆菌热休克蛋白65与人白细胞介素2的重组蛋白,为进一步研究其对结核疫苗的作用奠定基础。方法用PCR的方法分别从H37Rv DNA和质粒pGEM-Teasy-IL-2中扩增目的基因片段hsp65和IL-2,将各目的片段克隆至pMD18-T载体中进行测序,将测序正确的目的基因片段分别经EcoRI和ClaI,ClaI和HindⅢ双酶切后亚克隆至原核表达载体pPro-EX HTa,转化大肠杆菌DH5α,挑选阳性克隆,经IPTG诱导后进行融合表达,并通过镍柱对融合蛋白进行纯化。结果PCR法扩增获得的各目的基因片段与GenBank报道的一致。构建的融合蛋白原核表达载体在大肠杆菌中表达后,经SDS-PAGE和Western-blot分析,在Mr 78000处有特异性的蛋白表达条带。用镍柱进行亲和层析纯化后得到了高纯度的HSP65-IL-2融合蛋白。结论成功的构建了结核分枝杆菌HSP65与人IL-2的融合表达载体,并在大肠杆菌中获得高效表达,有望为结核的预防提供有效的疫苗。

To construct a recombinant plasmid carrying genes coding to Mycobacterium tuberculosis hsp 65 and human interleukin 2, and to express and purify this fusion protein for use in development of vaccine for tuberculosis, hsp65 gene was amplified by PCR with specific primers from the genome of Mycobacterium tuberculosis （MTB） H37Rv and IL-2 gene was amplified from plasmid pGEM-Teasy-IL-2. The PCR products were further inserted into pMD18-T vector for sequencing hsp65 and IL-2 genes, respectively. The EcoRI/ClaI restriction fragment of the hsp65 gene and the ClaI/Hindm fragment of the IL- 2 gene were ligated into expression vector pPro-EX HTa and transformed into E, coli DH5α. Then the expressed fusion protein of HSP65-IL-2 was purified by Ni-NTA system. Sequence of hsp65 and IL-2 by PCR amplified were identical with those of the GenBank reported. The result of SDS-PAGE showed that a fusion protein with relative molecular mass（Mr）of 78kD was expressed, which was confirmed by Western-blot analysis with specific monoclonal antibody against human IL-2. Thus,this purified fusion proteins obtained by Ni-NTA purification system. The fusion protein of HSP65-IL-2 constructed and expressed in E, coli DH5a successfully. It may be one of the useful candidate for the derelopment of tuberculosis vaccine.