【作者】 席丽；

【导师】 靳亚平；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2008， 硕士

【摘要】 结核分支杆菌(Mycobacterium tuberculosis, MTB)热休克蛋白6(5Heat Shock Protein 65, HSP65)是热休克蛋白60家族成员之一,由groEL2基因编码,是一种高度保守的蛋白质。该蛋白具有很强的免疫原性,在结核杆菌感染过程中,HSP65是机体对抗其入侵的重要的免疫保护性抗原之一,提示HSP65可能成为结核病预防和治疗的一个关键因素。本研究采用分子生物学方法分别构建MTB HSP65的原核重组质粒和真核重组质粒,测定分析了HSP65蛋白和真核重组质粒在小鼠体内的免疫学特性和抵抗MTB感染的保护力。1.采用PCR方法从MTB H37Rv株DNA基因组中扩增HSP65基因,与pMD18-T载体连接,酶切鉴定阳性重组质粒pMD-hsp65,经测序证实与Genebank公布的MTB HSP65基因序列完全一致。利用酶切位点将HSP65基因从pMD-hsp65中亚克隆入pPRO EX HTa原核表达载体,酶切鉴定阳性重组质粒pPRO-hsp65,并用IPTG进行诱导表达。对表达产物进行SDS-PAGE的结果表明,在相对分子量为65 Ku的位置有目的蛋白表达。用抗MTB HSP65的mAb及TB患者血清进行Western-blot鉴定,结果证实在相应的位置有特异性的结合条带。采用Ni-NTA亲和色谱柱在变性条件下纯化得到了HSP65蛋白。2.利用酶切位点将HSP65基因从pMD18-hsp65中亚克隆入真核表达载体pcDNA3.1(-),酶切鉴定阳性重组质粒pcDNA-hsp65。将重组质粒用阳离子聚合物法稳定转染P815(H-2d)细胞。分别经RT-PCR和间接免疫荧光法证实,该重组质粒可在稳定转染的P815细胞中转录和特异性地表达。经过G418压力筛选,得到8株能稳定表达HSP65蛋白的P815细胞系。3.用HSP65原核表达蛋白及真核重组质粒免疫BALB/c小鼠,并检测两种疫苗各项免疫学指标。结果表明,HSP65亚单位疫苗和基因疫苗均可在小鼠体内刺激产生较强的体液和细胞免疫应答,并各有特点;均可显著减少感染小鼠体内的脾荷菌数,表明其可抵抗MTB的感染,但这种保护作用均未超过BCG。上述结论为进一步研究HSP65疫苗的免疫学特性及与其他疫苗的优势组合,用于TB新型疫苗的开发提供了实验数据和物质基础。更多还原

【Abstract】 Mycobacterium tuberculosis (MTB) Heat Shock Protein 65(HSP65)which belongs to HSP60s,encoding by gene groEL2 ,is a kind of highly conservative protein. It is one of the important protective antigens for resistance of MTB infection. The protein has strong immunogenicity which has been proved that about 20% reactive T cell can discriminate HSP65 in MTB infected mouse, thus HSP65 may a key point for precaution and therapy of tuberculosis (TB).This research used the molecular biology method to construct prokaryotic recombinant plasmid and eukaryotic recombinant plasmid based on HSP65 of MTB .Compared with BCG, to evaluate the immunological properties and protective efficacy of them against MTB infection, in order to find a new efficient vaccine against TB. The results as follows:1. The gene of HSP65 was amplified from DNA genome of Mycobacterium tuberculosis H37Rv strain by polymerase chain reaction (PCR). Inserted the PCR product into pMD18-T vector and sequenced. Ligated the right sequenced HSP65 gene into pPRO EX HTa prokaryotic expression vector and identified by restrictive enzyme digestion, the recombinant plasmid was named pPRO-hsp65. The recombinant plasmid was transformed into E.coli DH5αstrain and induced with IPTG.The analysis of SDS-PAGE showed that there was a specific protein expression at 65Ku molecular marker, and the protein was further identified by Western-blot using anti- HSP65 mAb and serum of TB patient. Purified the fusion protein by Ni-NTA purification system.2. Subcloned the gene of HSP65 into eukaryotic expression vector pcDNA3.1 (-), the recombinant plasmid was named pcDNA-hsp65 and identified by restrictive enzyme digestion.Transfected the recombinant plasmid into P815 cells stably by Suohua-sofast transfection reagent. The specific mRNA of the gene was detected by RT-PCR, and the expression of HSP65 protein in the transfected cells was detected by indirect immunofluorescence technique (IIFT). The result showed that there were specific strap in the agarose gel electrophoresis (AGE) and green immunofluorescence showed in the transfected P815 cells. Eight lines of transfected P815 cells stably expressing the protein of HSP65 was selected by G418.3. Immunized BALB / c mice with prokaryotic expression protein and eukaryotic recombinant plasmid of HSP65 gene, and tested the two vaccines immunological parameters. Results showed that, both the subunit and DNA vaccine of HSP65 protein could elicite great humoral and cellular immune responses, and had their own characteristics. The two vaccines could significantly reduce the bacteria loads in spleen and lungs of infected mice, this meant that the vaccines could prevent the mice against MTB infection, but the ability of protection was not better than that of BCG.All these conclusions brought some new clues for researching prevention and treatment of tuberculosis.更多还原