構(gòu)建人巨細(xì)胞病毒多表位DNA疫苗的初步研究
發(fā)布時(shí)間：2018-08-13 10:00
【摘要】： 目的：人巨細(xì)胞病毒(human cytomegalovirus，hCMV)在人群中的感染率非常高，多為持續(xù)潛伏性感染，當(dāng)機(jī)體免疫力低下時(shí)發(fā)生嚴(yán)重疾病。臨床上尚無(wú)有效的治療方法，因此需要發(fā)展hCMV疫苗以促進(jìn)機(jī)體免疫力的恢復(fù)。常規(guī)的預(yù)防性疫苗對(duì)已感染的個(gè)體不能誘導(dǎo)有效的免疫應(yīng)答，因此必須研究開(kāi)發(fā)新型的治療性疫苗，清除病原體和異常細(xì)胞，使疾病得以治愈。表位是誘生特異性免疫反應(yīng)的最小功能單位，如能通過(guò)接種肽表位疫苗增加hCMV特異性活化CTL的數(shù)量，將能有效清除感染的細(xì)胞。但由于TCR識(shí)別的MHC限制性，單一的肽表位不能被大部分人群識(shí)別，為了發(fā)展適合大部分人群的多表位DNA疫苗，本實(shí)驗(yàn)聯(lián)合使用多種高頻HLA限制性的hCMV抗原表位構(gòu)建多表位的DNA真核表達(dá)質(zhì)粒，用其轉(zhuǎn)染HLA-A~*0201限制性hCMV陽(yáng)性個(gè)體的B細(xì)胞，并探討用其轉(zhuǎn)染的B細(xì)胞誘導(dǎo)hCMV特異性CTL活化的效能。為進(jìn)一步發(fā)展hCMV DNA疫苗積累資料，并為T(mén)細(xì)胞識(shí)別的數(shù)字化和可視化提供實(shí)驗(yàn)?zāi)Ｐ汀?方法：通過(guò)重疊PCR方法合成HLA-A2、HLA-A11和HLA-A24限性的六個(gè)hCMV抗原表位的DNA序列hCMV_(6epitopes)，通過(guò)雙酶切、連接插入到載體質(zhì)粒pVAX1中，構(gòu)建表達(dá)質(zhì)粒pVAX1-hCMV_(6epitopes)；為進(jìn)行細(xì)胞轉(zhuǎn)染和T細(xì)胞活化評(píng)價(jià)實(shí)驗(yàn)，在此基礎(chǔ)上，分別通過(guò)將PCR擴(kuò)增出的報(bào)告基因綠色熒光蛋白(GFP)DNA序列和hCMV_(6epitopes)DNA序列插入pVAX1-hCMV_(6epitopes)和pEGFP-N1，構(gòu)建質(zhì)粒pVAX1-hCMV_(6epitopes)-GFP和pEGFP-hCMV_(6epitopes)；通過(guò)酶切、PCR擴(kuò)增和DNA測(cè)序等方法鑒定質(zhì)粒構(gòu)建的正確性。將質(zhì)粒pVAX1-hCMV_(6epitopes)-GFP和pEGFP-hCMV_(6epitopes)通過(guò)脂質(zhì)體法分別轉(zhuǎn)染Hela細(xì)胞，檢驗(yàn)?zāi)康腄NA所編碼的蛋白在真核細(xì)胞內(nèi)的表達(dá)情況。通過(guò)核轉(zhuǎn)染儀將質(zhì)粒pEGFP-hCMV_(6epitopes)轉(zhuǎn)染HLA-A~*0201限制性hCMV陽(yáng)性個(gè)體的外周血B細(xì)胞，用流式細(xì)胞分析法分析pEGFP-hCMV_(6epitopes)在B細(xì)胞中的轉(zhuǎn)染率及hCMV特異性CTL的活化率。 結(jié)果：重疊PCR成功擴(kuò)增多表位的DNA序列hCMV_(6epitopes)，測(cè)序結(jié)果表明所構(gòu)建的三個(gè)質(zhì)粒序列都與預(yù)期相符，hCMW_(6epitopes)DNA和GFP DNA分別正確插入到載體質(zhì)粒中；重組質(zhì)粒pEGFP-hCMV_(6epitopes)和pVAX1-hCMV_(6epitopes)-GFP在Hela細(xì)胞內(nèi)能夠表達(dá)目的蛋白，呈綠色熒光；用pEGFP-hCMV_(6epitopes)轉(zhuǎn)染HLA-A~*0201限制性hCMV陽(yáng)性個(gè)體的外周血B細(xì)胞，轉(zhuǎn)染率僅為0.97％，但是這些B細(xì)胞已顯著誘導(dǎo)hCMV特異性CTL活化，CD69表達(dá)率達(dá)11.48％，對(duì)照組用pEGFP-N1轉(zhuǎn)染B細(xì)胞的轉(zhuǎn)染率為37.44％，但hCMV特異的CTL活化率僅為2.01％，，兩組有顯著差異。 結(jié)論：成功構(gòu)建重組質(zhì)粒pVAX1-hCMV_(6epitopes)、pVAX1-hCMV_(6epitopes)-GFP和pEGFP-hCMV_(6epitopes)，并且證明它們?cè)谡婧思?xì)胞內(nèi)能夠表達(dá)；pEGFP-hCMV_(6epitopes)轉(zhuǎn)染的HLA-A~*0201限制性個(gè)體的B淋巴細(xì)胞能夠正確表達(dá)、加工和提呈特異的hCMV抗原表位，誘導(dǎo)hCMV特異性T淋巴細(xì)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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392
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