【摘要】：在畢赤酵母中表達含有HCMV gp52 C末端，pp150 C末端以及gB C末端串聯(lián)的三合肽，并構(gòu)建其DNA疫苗，觀察該疫苗在新西蘭兔體內(nèi)的免疫效果。以期為檢測HCMV感染和HCMV疫苗的研制奠定實驗基礎(chǔ)。 構(gòu)建含有HCMV三合肽基因的重組質(zhì)粒pPIC9K-6His-HCMV3p，，并在畢赤酵母中表達重組三合肽，采用Ni~(2+)螯合層析法純化目的蛋白。將三合肽基因克隆入真核表達載體pVAX1中，構(gòu)建真核重組質(zhì)粒pVAX1-HCMV3p。對新西蘭兔進行肌肉免疫，ELISA法檢測兔血清中抗重組三合肽抗體水平，間接免疫熒光法檢測血清中HCMV結(jié)合抗體。并利用MTT法測定免疫兔的外周血淋巴細胞對ConA，PHA和HCMV刺激后的增殖指數(shù)，乳酸脫氫酶釋放法檢測免疫動物淋巴細胞對病毒的反應(yīng)。 SDS-PAGE和Western-blot結(jié)果表明在畢赤酵母中成功表達出重組三合肽蛋白，重組三合肽抗原可與兔抗HCMV血清發(fā)生特異性反應(yīng)。ELISA檢測結(jié)果表明DNA疫苗可在新西蘭兔體內(nèi)產(chǎn)生抗HCMV重組三合肽抗原的特異性抗體；間接免疫熒光結(jié)果顯示免疫兔血清也可與HCMV病毒顆粒發(fā)生特異性反應(yīng)；淋巴細胞增殖實驗顯示HCMV能特異性刺激免疫兔淋巴細胞增殖；免疫后的淋巴細胞可抵抗病毒的攻擊。 真核表達的人巨細胞病毒重組三合肽有可能作為抗原組分用于HCMV的檢測中；所構(gòu)建的疫苗可有效誘導(dǎo)新西蘭兔體液免疫和細胞免疫應(yīng)答，為人巨細胞病毒核酸疫苗的研究奠定基礎(chǔ)。
[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.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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相關(guān)期刊論文 前2條

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