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作 者:席丽[1] 师长宏[2] 靳亚平[1] 张海[2] 张廷芬[2] 赵勇[2] 岳晨莉[2]
机构地区:[1]西北农林科技大学动物医学院,陕西西安712100 [2]第四军医大学实验动物中心,陕西西安710033
出 处:《第四军医大学学报》2008年第9期769-772,共4页Journal of the Fourth Military Medical University
基 金:国家自然科学基金(30670116)
摘 要:目的:构建HSP65蛋白的亚单位疫苗和基因疫苗,并与BCG相比较,评价这两种疫苗在小鼠体内的免疫学特性.方法:从结核分枝杆菌H37Rv基因组中扩增出hsp65基因,连接进入pMD18-T载体中,测序后,利用不同酶切位点将完整的hsp65基因分别克隆到pProEX HTb原核表达载体和pcDNA3.1(-)真核表达载体,前者在大肠杆菌中诱导表达,经SDS-PAGE和Western Blot分析后,用Ni-NTA亲和层析柱进行纯化;后者通过阳离子聚合物瞬转P815细胞,用间接免疫荧光检测细胞内HSP65蛋白的表达.用上述原核表达蛋白和真核质粒(分别为亚单位疫苗和基因疫苗)免疫BALB/c小鼠,ELISA测定特异性抗体的滴度,MTT测定脾淋巴细胞特异性增殖指数.结果:获得的结核分枝杆菌hsp65基因序列与GenBank公布的完全一致;Western Blot结果显示,在Mr约65000处有与鼠抗HSP65 mAb和临床结核患者血清的特异性结合带;免疫荧光结果显示转染重组质粒的P815细胞的表达蛋白与HSP65 mAb有特异性结合.两种疫苗免疫小鼠体内产生特异性抗体,经蛋白刺激后可引起脾淋巴细胞特异性增殖.结论:所构建的两种疫苗能引起免疫动物的特异性体液和细胞免疫反应,应进一步研究其抵抗结核分支杆菌感染的保护力机制,以用于结核病的防治研究.AIM: To construct the subunit vaccine and DNA vaccine based on heat shock protein 65 ( HSP65 ) of M. tuberculosis H37Rv (MTB) and to evaluate the immunological properties. METHODS:The gene of hsp 65 was amplified from DNA genome of MTB H37Rv strain by polymerase chain reaction (PCR). The PCR product was inserted into pMD18-T vector and sequenced. The HSP65 gene segment was subeloned into prokaryotic expression vector pProEX HTb and eukaryotic expression vector pcD- NA3.1 ( - ) respectively. The former was expressed in E. coli DHSα. The target protein was identified by SDS-PAGE analysis and by Western Blot with monoclonal antibody against MTB HSP 65 and sera from clinical TB patients. Recombinant 6 x His fused protein was purified by Ni-NTA purification system; the eukaryotic recombinant plasmid was transfected into P815 cells by Suohuasofast transfection reagent, and the expression was identified by indirect immunofluorescent technology. RESULTS: The sequence of PCR product was identical with that of HSP65 gene in GenBank. Relative molecular mass identified by SDS-PAGE analysis was consistent with what had been reported, which was also confirmed by Western Blot analysis that there was a specific band at 65000 with the specific monoclonal antibody against MTB HSP65 and TB patients' sera, and there was a specific band between proteins expressed by transfected P815 and two antibodies above. CONCLUSION: The two vaccines of HSFK5 arc success filly constructed and possess the immunological properties. It will be helpful in further studies on diagnosis and therapy of tuberculosis.
关 键 词:结核分枝杆菌 热休克蛋白质类 亚单位疫苗 基因疫苗
分 类 号:R378.911[医药卫生—病原生物学]
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