【作者】 陈瑞；

【导师】 潘善培；

【作者基本信息】 暨南大学 ， 发育生物学， 2007， 硕士

【摘要】 目的：人巨细胞病毒（human cytomegalovirus，hCMV）在人群中的感染率非常高，多为持续潜伏性感染，当机体免疫力低下时发生严重疾病。临床上尚无有效的治疗方法，因此需要发展hCMV疫苗以促进机体免疫力的恢复。常规的预防性疫苗对已感染的个体不能诱导有效的免疫应答，因此必须研究开发新型的治疗性疫苗，清除病原体和异常细胞，使疾病得以治愈。表位是诱生特异性免疫反应的最小功能单位，如能通过接种肽表位疫苗增加hCMV特异性活化CTL的数量，将能有效清除感染的细胞。但由于TCR识别的MHC限制性，单一的肽表位不能被大部分人群识别，为了发展适合大部分人群的多表位DNA疫苗，本实验联合使用多种高频HLA限制性的hCMV抗原表位构建多表位的DNA真核表达质粒，用其转染HLA-A^*0201限制性hCMV阳性个体的B细胞，并探讨用其转染的B细胞诱导hCMV特异性CTL活化的效能。为进一步发展hCMV DNA疫苗积累资料，并为T细胞识别的数字化和可视化提供实验模型。方法：通过重叠PCR方法合成HLA-A2、HLA-A11和HLA-A24限性的六个hCMV抗原表位的DNA序列hCMV_6epitopes，通过双酶切、连接插入到载体质粒pVAX1中，构建表达质粒pVAX1-hCMV_6epitopes；为进行细胞转染和T细胞活化评价实验，在此基础上，分别通过将PCR扩增出的报告基因绿色荧光蛋白（GFP）DNA序列和hCMV_6epitopesDNA序列插入pVAX1-hCMV_6epitopes和pEGFP-N1，构建质粒pVAX1-hCMV_6epitopes-GFP和pEGFP-hCMV_6epitopes；通过酶切、PCR扩增和DNA测序等方法鉴定质粒构建的正确性。将质粒pVAX1-hCMV_6epitopes-GFP和pEGFP-hCMV_6epitopes通过脂质体法分别转染Hela细胞，检验目的DNA所编码的蛋白在真核细胞内的表达情况。通过核转染仪将质粒pEGFP-hCMV_6epitopes转染HLA-A^*0201限制性hCMV阳性个体的外周血B细胞，用流式细胞分析法分析pEGFP-hCMV_6epitopes在B细胞中的转染率及hCMV特异性CTL的活化率。结果：重叠PCR成功扩增多表位的DNA序列hCMV_6epitopes，测序结果表明所构建的三个质粒序列都与预期相符，hCMW_6epitopesDNA和GFP DNA分别正确插入到载体质粒中；重组质粒pEGFP-hCMV_6epitopes和pVAX1-hCMV_6epitopes-GFP在Hela细胞内能够表达目的蛋白，呈绿色荧光；用pEGFP-hCMV_6epitopes转染HLA-A^*0201限制性hCMV阳性个体的外周血B细胞，转染率仅为0.97％，但是这些B细胞已显著诱导hCMV特异性CTL活化，CD69表达率达11.48％，对照组用pEGFP-N1转染B细胞的转染率为37.44％，但hCMV特异的CTL活化率仅为2.01％，两组有显著差异。结论：成功构建重组质粒pVAX1-hCMV_6epitopes、pVAX1-hCMV_6epitopes-GFP和pEGFP-hCMV_6epitopes，并且证明它们在真核细胞内能够表达；pEGFP-hCMV_6epitopes转染的HLA-A^*0201限制性个体的B淋巴细胞能够正确表达、加工和提呈特异的hCMV抗原表位，诱导hCMV特异性T淋巴细胞的活化。更多还原

【Abstract】 Aim: The rate of hCMV infection is very high. HCMV generally persistents latent in thebody, however, active infection occurs when the immunity of organism descends, and thencauses severe diseases. So far there are not any effective therapies. It is necessary to developvaccines which can restore the immunity of organism. Traditional prophylactic vaccines can notinduce effective immune responses in the infected individuals, it is imperative to develop noveltherapeutic vaccines to sufficiently induce CD8⁺T cell immunity so as to clear up the hCMVlatent in the organism. Epitope is the minimal functional unit to induce immune response, if thenumber of hCMV specific T cells can be augmented by peptide epitope vaccines, the hCMVlatent in the organism can be eliminated. Because of restriction of TCR recognition, one singlepeptide epitope can not be recognized by a majority of population. To develop a multi-epitopevaccine which can be used in most population, we combined six epitopes restricted by highfrequency HLAs to construct multi-epitope DNA eukaryotic expressing plasmids, thentransfected B cells in the PBMCs of hCMV positive and HLA-A*0201-restricted individual withthe multi-epitope DNA recombinant plasmid and analyzed the activation of hCMV specific Tcells. In this study we provided primary data for the further development of hCMV vaccines andexperimental models for the digital and visual recognition of T cell.Methods: The DNA sequence of hCMV_6epitopes, which include HLA-A2, All andA24-restricted epitopes was synthesized through overlap PCR, and inserted into the vectorplasmid pVAX1 to construct eukaryotic expressing plasmid. For the studies of evaluatinghCMV_6epitopes expression of transfected cells and the activation of T cells, pEGFP-hCMV_6epitopesand pVAX1-hCMV_6epitopes-GFP were constructed by inserting hCMV_6epitopes and the reportinggene GFP DNA sequence into vector plasmids pEGFP-N1 and pVAXl-hCMV_6epitopesrespectively. Three plasmids mentioned above were identified by PCR identifications,dual-enzyme digest, gel electrophoresis and sequencing. Hela cells were transfected respectivelywith plasmids pEGFP-hCMV_6epitopes and pVAX1-hCMV_6epitopes-GFP by lipofectamine to test theexpression of protein encoded by target sequence in eukaryotic cells. The B cells in theindividual of hCMV positive and HLA-A*0201-restricted were transfected with plasmidpEGFP-hCMV_6epitopes by Nucleofetor TM device, the transfection rate in the B cells and the activation rate of hCMV specific T cells were analyzed by FCS.Results: The multi-epitope DNA sequence hCMV_6epitopes had been combined successfully.pVAX1-hCMV_6epitopes, pVAX1-hCMV_6epitopes-GFP and pEGFP-hCMV_6epitopes were constructedcorrectly, hCMV_6epitopes DNA and GFP DNA respectively inserted into plasmid vectors correctly.Recombinant plasmids pVAX1-hCMV_6epitopes and pEGFP-hCMV_6epitopes were able to expresstarget proteins in Hela cells, showing green fluorescent. When the B cells in the PBMCs ofhCMV positive and HLA-A*0201-restricted individual were transfected with recombinantplasmid pEGFP-hCMV_6epitopes, tranfecting rate was only 0.97%. But they were able to inducerecognitions and activations of hCMV specific CTLs, with CD69 expression rate reaching11.48%. While in the control group, the tranfection rate of pEGFP-N1 in B cells was 37.44%,and the activation rate of hCMV specific CTL was only 2.01%. It showed differencesignificantly between the two groups.Conclusions: hCMV_6epitopes DNA was synthesized successfully. Recombinant plasmidspVAX1-hCMV_6epitopes, pVAX1-hCMV_6epitopes-GFP and pEGFP-hCMV_6epitopes were constructedsuccessfully and the target proteins could be expressed in the Hala cells. The B cells in thePBMCs of hCMV positive and HLA-A*0201-restricted individual transfected with plasmidpEGFP-hCMV_6epitopes could correctly express, process and present hCMV specific antigenepitopes, and could induce the activations of hCMV specific T cells.更多还原