细粒棘球蚴（中国大陆株）热休克蛋白70重组疫苗的研究

发布时间：2019-06-20 20:01

【摘要】： 目的克隆细粒棘球蚴(Echinococcus granulosus,Eg)热休克蛋白70(Heat shock protein70,HSP70)基因,构建重组原核表达质粒,表达并纯化重组EgHSP70,对该重组蛋白进行免疫学特性鉴定,为筛选有价值的包虫病候选疫苗分子奠定基础。方法①利用生物信息学原理,在互联网中检索获得EgHSP70核苷酸序列,以此作为侯选基因;从细粒棘球蚴原头蚴中提取总RNA,通过RT-PCR的方法扩增出EgHSP70基因,纯化PCR产物,将其重组到pGEM-T载体后进行序列测定和特性分析。②将测序结果输入DNAStar分析软件和互联网SWISS-MODEL数据库,对重组EgHSP70的特性、抗原表位及构象进行预测分析。③构建HSP70 /pGEX-6P-1/BL21工程菌株并对其进行鉴定,诱导表达融合蛋白并通过SDS-PAGE鉴定。亲和层析法纯化重组融合蛋白HSP70/GST和重组HSP70,用实验室已有的细粒棘球蚴囊壁免疫的兔抗血清通对其进行初步的免疫学鉴定。④用HSP70/GST和HSP70分别免疫小鼠,制备抗血清。通过Western blot和ELISA对重组EgHSP70的免疫学特性进行鉴定。结果①RT-PCR成功扩增EgHSP70目的基因,并成功构建HSP70/pGEM-T/JM109工程菌株,经测序表明该片段由402bp组成,其开放阅读框完整无误。同源性分析结果表明,与原检索序列相比,有14个碱基不同,同源性达96%,推导编码氨基酸序列同源性为81%。②DNAStar分析软件推算氨基酸序列结果显示,EgHSP70由133个理论氨基酸组成,分子量约为14.53kDa。DNAStar分析软件确定了重组EgHSP70可能的抗原表位。与其他动物热休克蛋白70氨基酸的同源性分析结果显示具有高度的保守性。③成功构建HSP70/pGEX-6P-1/BL21重组表达质粒,表达后获得融合蛋白HSP70/GST分子量约为40kDa,重组EgHSP70分子量约为14kDa。Western blot证实重组EgHSP70能被免疫兔血清识别。④Western blot显示免疫小鼠血清能够有效识别重组蛋白;ELISA法检测结果表明,原头蚴、囊液等天然抗原均可被免疫小鼠血清识别,与对照组相比有显著的差异(P0.05)。提示,重组抗原可诱导免疫小鼠产生特异性抗体。结论①成功克隆出EgHSP70重组抗原候选基因。②成功构建高效融合表达基因工程菌株HSP70/pGEX-6P-1 /BL21。③动物实验表明,重组EgHSP70具有较好的免疫原性和抗原性,可望作为包虫病疫苗侯选分子,值得进一步深入研究。④经文献检索,国内尚未报道有关EgHSP70基因的克隆及重组蛋白的表达、纯化及免疫学特性鉴定的研究。
[Abstract]:Objective to clone the heat shock protein 70 (Heat shock protein70,HSP70 gene of Echinococcus granulosus (Echinococcus granulosus,Eg), construct the recombinant prokaryotic expression plasmid, express and purify the recombinant EgHSP70, to identify the immunological characteristics of the recombinant protein, and lay a foundation for screening valuable candidate vaccine molecules for echinococcosis. Methods 1 according to the principle of bioinformatics, EgHSP70 nucleotides were obtained from the Internet as candidate genes. The total RNA, was extracted from Echinococcus granulosus. The EgHSP70 gene was amplified by RT-PCR, the PCR product was purified and recombined into pGEM-T vector for sequencing and characterization. 2 the sequencing results were input into DNAStar analysis software and Internet SWISS-MODEL database to predict and analyze the characteristics, antigenic epitopes and conformations of the recombinant EgHSP70. 3 the HSP70 / pGEX-6P-1/BL21 engineering strain was constructed and identified. The fusion protein was induced and identified by SDS-PAGE. Purification of recombinant fusion protein HSP70/GST by affinity chromatography and recombinant HSP70, were identified by rabbit antiserum Tong immunized with Echinococcus granulosus cyst wall in laboratory. 4 mice were immunized with HSP70/GST and HSP70 respectively to prepare antiserum. The immunological characteristics of recombinant EgHSP70 were identified by Western blot and ELISA. Results the EgHSP70 target gene was successfully amplified by 1RT-PCR and the HSP70/pGEM-T/JM109 engineering strain was successfully constructed. the sequencing results showed that the fragment was composed of 402bp and its open reading frame was complete and correct. The results of homology analysis showed that compared with the original retrieval sequence, there were 14 bases with 96% homology. The deduced amino acid sequence homology was calculated by 81%.2DNAStar analysis software. The results showed that EgHSP70 was composed of 133 theoretical amino acids, and the molecular weight was about the possible antigenic epitope of recombinant EgHSP70 determined by 14.53kDa.DNAStar analysis software. The homology analysis of amino acids with heat shock protein 70 in other animals showed that it was highly conserved. 3 the recombinant expression plasmid HSP70/pGEX-6P-1/BL21 was successfully constructed and the molecular weight of fusion protein HSP70/GST was about 40 KDa. the molecular weight of recombinant EgHSP70 was about 14kDa.Western blot, which confirmed that the recombinant EgHSP70 could be recognized by immunized rabbit serum. 4 Western blot showed that the recombinant protein could be effectively recognized in immunized mouse serum. The results of ELISA showed that the natural antigens such as procephalus and cysticercus could be recognized by the serum of immunized mice, which were significantly different from those of the control group (P 0.05). It is suggested that the recombinant antigen can induce the production of specific antibodies in immunized mice. Conclusion 1 the candidate gene .2 of EgHSP70 recombinant antigen was successfully cloned and the highly efficient fusion expression gene engineering strain HSP70/pGEX-6P-1 / BL21.3 was successfully constructed. The results showed that the recombinant EgHSP70 had good immunogenicity and antigenicity and was expected to be used as a candidate molecule for echinococcosis vaccine. 4 the cloning of EgHSP70 gene and the expression of recombinant protein have not been reported in China. Study on purification and immunological characterization.
【学位授予单位】：宁夏医学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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