人巨细胞病毒三合肽的表达及DNA疫苗的研制

发布时间：2019-05-14 05:08

【摘要】：在毕赤酵母中表达含有HCMV gp52 C末端，pp150 C末端以及gB C末端串联的三合肽，并构建其DNA疫苗，观察该疫苗在新西兰兔体内的免疫效果。以期为检测HCMV感染和HCMV疫苗的研制奠定实验基础。构建含有HCMV三合肽基因的重组质粒pPIC9K-6His-HCMV3p，，并在毕赤酵母中表达重组三合肽，采用Ni~(2+)螯合层析法纯化目的蛋白。将三合肽基因克隆入真核表达载体pVAX1中，构建真核重组质粒pVAX1-HCMV3p。对新西兰兔进行肌肉免疫，ELISA法检测兔血清中抗重组三合肽抗体水平，间接免疫荧光法检测血清中HCMV结合抗体。并利用MTT法测定免疫兔的外周血淋巴细胞对ConA，PHA和HCMV刺激后的增殖指数，乳酸脱氢酶释放法检测免疫动物淋巴细胞对病毒的反应。 SDS-PAGE和Western-blot结果表明在毕赤酵母中成功表达出重组三合肽蛋白，重组三合肽抗原可与兔抗HCMV血清发生特异性反应。ELISA检测结果表明DNA疫苗可在新西兰兔体内产生抗HCMV重组三合肽抗原的特异性抗体；间接免疫荧光结果显示免疫兔血清也可与HCMV病毒颗粒发生特异性反应；淋巴细胞增殖实验显示HCMV能特异性刺激免疫兔淋巴细胞增殖；免疫后的淋巴细胞可抵抗病毒的攻击。真核表达的人巨细胞病毒重组三合肽有可能作为抗原组分用于HCMV的检测中；所构建的疫苗可有效诱导新西兰兔体液免疫和细胞免疫应答，为人巨细胞病毒核酸疫苗的研究奠定基础。
[Abstract]:Tripeptide containing HCMV gp52 C terminal, pp150 C terminal and gB C terminal was expressed in Pichia pastoris, and its DNA vaccine was constructed to observe the immune effect of the vaccine in New Zealand rabbits. In order to lay an experimental foundation for the detection of HCMV infection and the development of HCMV vaccine. The recombinant plasmid pPIC9K-6His-HCMV3p, containing HCMV tripeptide gene was constructed and expressed in Pichia pastoris. The target protein was purified by Ni~ (2) chelating chromatography. The triad peptide gene was cloned into eukaryotic expression vector pVAX1 to construct eukaryotic recombinant plasmid pVAX1-HCMV3p.. Muscle immunization was performed in New Zealand rabbits. ELISA assay was used to detect the level of anti-recombinant triad peptide antibody in rabbit serum, and indirect immunofluorescence assay was used to detect HCMV binding antibody in serum. The proliferation index of peripheral blood lymphocytes stimulated by ConA,PHA and HCMV was measured by MTT assay, and the response of immunized animal lymphocytes to virus was detected by lactic dehydrogenase release assay. The results of SDS-PAGE and Western-blot showed that the recombinant tripeptide protein was successfully expressed in Pichia pastoris. The recombinant tripeptide antigen could react specifically with rabbit anti-HCMV serum. Elisa results showed that DNA vaccine could produce specific antibody against HCMV recombinant tripeptide antigen in New Zealand rabbits. The results of indirect immunofluorescence showed that the immunized rabbit serum could also react specifically with HCMV virus particles, the lymphocyte proliferation test showed that HCMV could specifically stimulate the proliferation of immunized rabbit lymphocytes, and the immunized lymphocytes could resist the attack of the virus. The eukaryotic expression of recombinant tripeptide of human cytomegalovirus may be used as an antigen component in the detection of HCMV. The constructed vaccine can effectively induce humoral and cellular immune responses in New Zealand rabbits, which lays a foundation for the study of human cytomegalovirus nucleic acid vaccine.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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