构建人巨细胞病毒多表位DNA疫苗的初步研究
发布时间：2018-08-13 10:00
【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_(6epitopes)DNA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_(6epitopes)DNA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。
[Abstract]:Objective: the infection rate of human cytomegalovirus (hCMV) in the population is very high. There is no effective treatment in clinic, so we need to develop hCMV vaccine to promote immune recovery. Conventional prophylactic vaccines cannot induce an effective immune response to infected individuals, so it is necessary to develop new therapeutic vaccines to remove pathogens and abnormal cells so that the disease can be cured. Epitopes are the smallest functional units to induce specific immune responses. If the number of hCMV specific activated CTL can be increased by inoculating peptide epitope vaccines, infected cells will be effectively eliminated. However, because of the restriction of MHC recognized by TCR, a single peptide epitope cannot be recognized by most people. In order to develop a multiepitope DNA vaccine suitable for most people, In this study, the eukaryotic expression plasmid of DNA was constructed by using a variety of high frequency HLA restricted hCMV epitopes. The eukaryotic expression plasmid was transfected into B cells of HLA-A~*0201 restricted hCMV positive individuals, and the efficiency of hCMV specific CTL activation induced by B cells was investigated. In order to further develop hCMV DNA vaccine accumulation data and provide experimental model for T cell recognition digitization and visualization. Methods: the hCMV _ (6epitopes) sequences of HLA-A2T HLA-A11 and six hCMV epitopes of HLA-A24 were synthesized by overlapping PCR method, and inserted into the vector pVAX1 by double enzyme digestion to construct the expression plasmid pVAX1-hCMV _ (6epitopes) for the purpose of cell transfection and T cell activation evaluation, the expression plasmid pVAX1-hCMV _ (6epitopes) was constructed by double restriction endonuclease digestion, and the expression plasmid pVAX1-hCMV _ (6epitopes) was constructed. On this basis, the reporter gene (GFP) DNA sequence and hCMV _ (6epitopes) DNA sequence amplified by PCR were inserted into pVAX1-hCMVN _ (6epitopes) and pEGFP-N1 respectively to construct pVAX1-hCMV_ (6epitopes) -GFP and pEGFP-hCMV_ (6epitopes). The plasmid pVAX1-hCMV-GFP and pEGFP-hCMVGFP were transfected into Hela cells by liposome method, respectively. The expression of the protein encoded by DNA in eukaryotic cells was examined. The plasmid pEGFP-hCMV _ (6epitopes) was transfected into peripheral blood B cells of HLA-A~*0201 restricted hCMV positive individuals by nuclear transfection instrument. The transfection rate of pEGFP-hCMV _ (6epitopes) in B cells and the activation rate of hCMV specific CTL were analyzed by flow cytometry. Results: the multiepitope DNA sequence hCMV _ (6epitopes) was successfully amplified by overlapping PCR. The results showed that the three plasmid sequences were all correctly inserted into the vector plasmids, respectively, in accordance with the expected sequence of hCMW _ (6epitopes) DNA and GFP DNA. The recombinant plasmids pEGFP-hCMV _ (6epitopes) and pVAX1-hCMV _ (6epitopes) -GFP can express the target protein and present green fluorescence in Hela cells, and the transfection efficiency of pEGFP-hCMVV _ (6epitopes) to B cells in peripheral blood of HLA-A~*0201 restricted hCMV positive individuals is only 0.97. However, these B cells had significantly induced hCMV specific CTL activation and CD69 expression rate was 11.48%. In the control group, the transfection rate of B cells transfected with pEGFP-N1 was 37.44%, but the hCMV specific CTL activation rate was only 2.01g, there was significant difference between the two groups. Conclusion: the recombinant plasmids pVAX1-hCMV _ (6epitopes) -GFP and pEGFP-hCMV _ (6epitopes) were successfully constructed, and it was proved that the B lymphocytes of HLA-A~*0201 restricted individuals transfected with pEGFP-hCMV _ (6epitopes) could correctly express, process and present specific hCMV epitopes. To induce the activation of hCMV specific T lymphocytes.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2180644
[Abstract]:Objective: the infection rate of human cytomegalovirus (hCMV) in the population is very high. There is no effective treatment in clinic, so we need to develop hCMV vaccine to promote immune recovery. Conventional prophylactic vaccines cannot induce an effective immune response to infected individuals, so it is necessary to develop new therapeutic vaccines to remove pathogens and abnormal cells so that the disease can be cured. Epitopes are the smallest functional units to induce specific immune responses. If the number of hCMV specific activated CTL can be increased by inoculating peptide epitope vaccines, infected cells will be effectively eliminated. However, because of the restriction of MHC recognized by TCR, a single peptide epitope cannot be recognized by most people. In order to develop a multiepitope DNA vaccine suitable for most people, In this study, the eukaryotic expression plasmid of DNA was constructed by using a variety of high frequency HLA restricted hCMV epitopes. The eukaryotic expression plasmid was transfected into B cells of HLA-A~*0201 restricted hCMV positive individuals, and the efficiency of hCMV specific CTL activation induced by B cells was investigated. In order to further develop hCMV DNA vaccine accumulation data and provide experimental model for T cell recognition digitization and visualization. Methods: the hCMV _ (6epitopes) sequences of HLA-A2T HLA-A11 and six hCMV epitopes of HLA-A24 were synthesized by overlapping PCR method, and inserted into the vector pVAX1 by double enzyme digestion to construct the expression plasmid pVAX1-hCMV _ (6epitopes) for the purpose of cell transfection and T cell activation evaluation, the expression plasmid pVAX1-hCMV _ (6epitopes) was constructed by double restriction endonuclease digestion, and the expression plasmid pVAX1-hCMV _ (6epitopes) was constructed. On this basis, the reporter gene (GFP) DNA sequence and hCMV _ (6epitopes) DNA sequence amplified by PCR were inserted into pVAX1-hCMVN _ (6epitopes) and pEGFP-N1 respectively to construct pVAX1-hCMV_ (6epitopes) -GFP and pEGFP-hCMV_ (6epitopes). The plasmid pVAX1-hCMV-GFP and pEGFP-hCMVGFP were transfected into Hela cells by liposome method, respectively. The expression of the protein encoded by DNA in eukaryotic cells was examined. The plasmid pEGFP-hCMV _ (6epitopes) was transfected into peripheral blood B cells of HLA-A~*0201 restricted hCMV positive individuals by nuclear transfection instrument. The transfection rate of pEGFP-hCMV _ (6epitopes) in B cells and the activation rate of hCMV specific CTL were analyzed by flow cytometry. Results: the multiepitope DNA sequence hCMV _ (6epitopes) was successfully amplified by overlapping PCR. The results showed that the three plasmid sequences were all correctly inserted into the vector plasmids, respectively, in accordance with the expected sequence of hCMW _ (6epitopes) DNA and GFP DNA. The recombinant plasmids pEGFP-hCMV _ (6epitopes) and pVAX1-hCMV _ (6epitopes) -GFP can express the target protein and present green fluorescence in Hela cells, and the transfection efficiency of pEGFP-hCMVV _ (6epitopes) to B cells in peripheral blood of HLA-A~*0201 restricted hCMV positive individuals is only 0.97. However, these B cells had significantly induced hCMV specific CTL activation and CD69 expression rate was 11.48%. In the control group, the transfection rate of B cells transfected with pEGFP-N1 was 37.44%, but the hCMV specific CTL activation rate was only 2.01g, there was significant difference between the two groups. Conclusion: the recombinant plasmids pVAX1-hCMV _ (6epitopes) -GFP and pEGFP-hCMV _ (6epitopes) were successfully constructed, and it was proved that the B lymphocytes of HLA-A~*0201 restricted individuals transfected with pEGFP-hCMV _ (6epitopes) could correctly express, process and present specific hCMV epitopes. To induce the activation of hCMV specific T lymphocytes.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前5条
1 赵缜;移植病人中HCMV激活感染标志的研究进展[J];国外医学.病毒学分册;2001年06期
2 张明生,黄华梁;抗原提呈的研究进展[J];国外医学(免疫学分册);2000年04期
3 李杰,王立顺,李一;T细胞表位疫苗的种类及其生物学意义[J];国外医学(免疫学分册);2000年04期
4 李夏,陈素文,喻达辉;绿色荧光蛋白及其在转基因动物研究中的应用[J];南方水产;2005年05期
5 何贤辉,徐丽慧,刘毅,蔡小嫦,曾耀英;HLA-A0201~*与EGFP融合蛋白表达载体的构建及其在K562细胞的表达和定位[J];中国病理生理杂志;2004年05期
本文编号：2180644
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2180644.html
最近更新
教材专著
