本文選題：乙型肝炎病毒 + 復(fù)制子DNA疫苗　； 參考：《第四軍醫(yī)大學(xué)》2013年碩士論文

【摘要】：研究背景： 乙型肝炎病毒（Hepatitis B virus, HBV）慢性感染多年來(lái)已成為影響公共健康的世界性難題之一，每年大約有100萬(wàn)人死于HBV慢性感染所致的肝硬化、肝衰竭和肝細(xì)胞癌。迄今為止，HBV慢性感染尚無(wú)有效治療手段，通過(guò)治療性疫苗激發(fā)機(jī)體自身免疫是治療慢性乙肝的重要途徑。課題組在治療性疫苗領(lǐng)域多年研究探索，自主研發(fā)的新型復(fù)制子DNA疫苗載體系統(tǒng)pSVK，能在常規(guī)DNA疫苗1/100的劑量即可激發(fā)高效的細(xì)胞免疫應(yīng)答。針對(duì)慢性乙肝治療中的核心難題“免疫耐受”，實(shí)驗(yàn)室前期構(gòu)建了融合多種免疫增效策略的新型復(fù)制子DNA疫苗pSVK-HBVA，使其在靶抗原攝入、表達(dá)、呈遞和免疫應(yīng)答激活等多個(gè)方面具備突出優(yōu)勢(shì)，有利于打破慢性乙肝的免疫耐受。本研究擬開(kāi)展乙肝新型治療性疫苗pSVK-HBVA免疫小鼠后的細(xì)胞免疫應(yīng)答研究，同時(shí)為檢測(cè)新型疫苗pSVK-HBVA的細(xì)胞毒T淋巴細(xì)胞（cytotoxicity Tlymphocyte,CTL）活性，還構(gòu)建了融合HBV多種抗原基因的重組表達(dá)質(zhì)粒pIRES-neo-HBAg。 研究目的： 開(kāi)展新型復(fù)制子DNA疫苗pSVK-HBVA免疫小鼠后的細(xì)胞免疫應(yīng)答研究，進(jìn)一步驗(yàn)證經(jīng)過(guò)疫苗優(yōu)化設(shè)計(jì)后的新型治療性復(fù)制子DNA疫苗的免疫治療功效。此外，還構(gòu)建了包含HBV多個(gè)抗原區(qū)段的真核表達(dá)質(zhì)粒pIRES-neo-HBAg，初步篩選了混合克隆細(xì)胞株，為進(jìn)一步篩選穩(wěn)定表達(dá)HBV融合抗原的單克隆細(xì)胞株奠定實(shí)驗(yàn)基礎(chǔ)。 研究方法： 首先，以本研究室前期構(gòu)建的HBV新型治療性復(fù)制子DNA疫苗pSVK-HBVA免疫BALB/c小鼠，末次免疫后一周進(jìn)行分離小鼠脾臟淋巴細(xì)胞，通過(guò)IFN-γ釋放的酶聯(lián)免疫斑點(diǎn)（ELISPOT）法檢測(cè)免疫后小鼠的CD8+T細(xì)胞活性，CCK-8（Cell Counting Kit-8）法檢測(cè)淋巴細(xì)胞在特異性乙肝抗原刺激下的免疫增殖活性，ELISA法檢測(cè)脾淋巴細(xì)胞免疫相關(guān)細(xì)胞因子包括Th1類因子IL-2、IFN-γ和Th2類因子IL-4、IL-10的免疫釋放；其次，為進(jìn)一步研究該疫苗激活的CTL免疫反應(yīng)，以pVAX1-HBVA為模板，設(shè)計(jì)引物進(jìn)行PCR擴(kuò)增包含HBV前S1、前S2、HBsAg和HBcAg的融合抗原基因（命名為HBAg），克隆入真核表達(dá)載體pIRES-neo中，構(gòu)建重組表達(dá)質(zhì)粒pIRES-neo-HBAg，并進(jìn)行酶切和測(cè)序驗(yàn)證；最后將重組質(zhì)粒pIRES-neo-HBAg進(jìn)行脂質(zhì)體法瞬時(shí)轉(zhuǎn)染293T細(xì)胞，轉(zhuǎn)染后48h通過(guò)Western blot、流式細(xì)胞術(shù)及免疫熒光等方法檢測(cè)和分析其表達(dá)情況，然后,再將提取的pIRES-neo-HBAg重組質(zhì)粒轉(zhuǎn)染Hepa1-6細(xì)胞（BALB/c小鼠來(lái)源的肝癌細(xì)胞），確定G418的最佳篩選濃度，加壓篩選后獲得陽(yáng)性混合克隆，流式細(xì)胞術(shù)檢測(cè)HBV融合抗原的表達(dá)。 研究結(jié)果： HBV新型治療性復(fù)制子DNA疫苗pSVK-HBVA，在免疫劑量?jī)H為1μg時(shí)就表現(xiàn)出很強(qiáng)的細(xì)胞免疫學(xué)活性。在特異性抗原刺激下疫苗免疫組小鼠淋巴細(xì)胞IFN-γ因子釋放顯著，CD8+T細(xì)胞被免疫激活；CCK-8檢測(cè)顯示疫苗免疫組小鼠的脾淋巴細(xì)胞顯示出很強(qiáng)的增殖活性；細(xì)胞因子釋放的ELISA檢測(cè)顯示，疫苗免疫后激活的細(xì)胞免疫反應(yīng)向Th1類反應(yīng)偏移。此外，重組質(zhì)粒pIRES-neo-HBAg經(jīng)酶切鑒定和測(cè)序分析，證實(shí)構(gòu)建成功；瞬時(shí)轉(zhuǎn)染293T細(xì)胞后，經(jīng)Western blot檢測(cè)顯示， HBV融合基因得到表達(dá)，，且分子量大小正確；流式細(xì)胞術(shù)檢測(cè)顯示，表達(dá)融合抗原HBAg的陽(yáng)性細(xì)胞率為20.43%；免疫熒光檢測(cè)結(jié)果顯示重組質(zhì)粒pIRES-neo-HBAg轉(zhuǎn)染細(xì)胞顯示紅色熒光，獲得有效表達(dá)。隨后，將重組質(zhì)粒pIRES-neo-HBAg轉(zhuǎn)染Hepa1-6細(xì)胞，并用800μg/ml的G418加壓篩選，初步篩選獲得了穩(wěn)定表達(dá)HBV融合抗原的混合克隆細(xì)胞，流式細(xì)胞術(shù)檢測(cè)顯示HBV融合抗原基因的陽(yáng)性細(xì)胞率可達(dá)86.20%。 研究結(jié)論： 新型治療性復(fù)制子DNA疫苗pSVK-HBVA在免疫劑量較低的情況下即可激活高效的細(xì)胞免疫應(yīng)答，且激活的免疫反應(yīng)向Th1類應(yīng)答偏移，有利于改善常規(guī)DNA免疫原性不足的問(wèn)題，也有利于克服慢性乙肝的免疫耐受。此外，本研究還構(gòu)建了表達(dá)乙肝多種復(fù)合抗原的真核表達(dá)質(zhì)粒pIRES-neo-HBAg，初步篩選了混合克隆細(xì)胞株，為建立有效的HBV體外細(xì)胞模型和方便的荷瘤小鼠動(dòng)物模型奠定了基礎(chǔ)。
[Abstract]:Research background:
Chronic hepatitis B virus (Hepatitis B virus, HBV) chronic infection has become one of the world's problems affecting public health for many years. About 1 million people die of liver cirrhosis, liver failure and hepatocellular carcinoma caused by chronic HBV infection every year. So far, there is no effective treatment for HBV chronic infection. Pestilence is an important way to treat chronic hepatitis B. In the field of therapeutic vaccine for many years, the new replicon DNA vaccine carrier system, pSVK, can stimulate efficient cellular immune response at the dose of conventional DNA vaccine 1/100. "Immune tolerance", the core problem in the treatment of chronic hepatitis B, in the early laboratory A new type of replicon DNA vaccine pSVK-HBVA, which combines multiple immunization strategies, has been constructed to make it have a prominent advantage in many aspects such as target antigen intake, expression, presentation and immune response activation, which is beneficial to break the immune tolerance of chronic hepatitis B. This study aims to develop a new therapeutic vaccine of hepatitis B for pSVK-HBVA immunization in mice. In response to the study, in order to detect the cytotoxic T lymphocyte (cytotoxicity Tlymphocyte, CTL) activity of the new vaccine pSVK-HBVA, the recombinant expression plasmid pIRES-neo-HBAg., which fused multiple antigen genes of HBV, was also constructed.
The purpose of the study is:
The immune response of the new-type replicon DNA vaccine pSVK-HBVA immunized mice was carried out to further verify the immunotherapy efficacy of the new therapeutic replicator DNA vaccine after the vaccine optimization. In addition, the eukaryotic expression plasmid pIRES-neo-HBAg containing multiple antigen segments of HBV was constructed, and the mixed cloned cells were screened preliminarily. These results provide an experimental basis for further screening of monoclonal cell lines expressing HBV fusion antigen.
Research methods:
First, BALB/c mice were immunized with a new HBV therapeutic replicator DNA vaccine pSVK-HBVA, which was constructed in the early stage of the study. The spleen lymphocytes were isolated from the mice one week after the last immunization. The activity of CD8+T cells in the immune mice was detected by the enzyme linked immunosorbent assay (ELISPOT) released by IFN- gamma, and the CCK-8 (Cell Counting Kit-8) method was used to detect the lymphocytes. The immuno proliferative activity of the specific hepatitis B antigen stimulated by the ELISA method was used to detect the immune related cytokines of the spleen lymphocyte, including the Th1 class factor IL-2, IFN- gamma and Th2 class factor IL-4, and the immune release of IL-10. Secondly, the CTL immune response activated by the vaccine was further studied, and pVAX1-HBVA was used as a template for PCR amplification containing HBV. The fusion antigen gene of anterior S1, pre S2, HBsAg and HBcAg (named HBAg) was cloned into the eukaryotic expression vector pIRES-neo, and the recombinant expression plasmid pIRES-neo-HBAg was constructed and verified by enzyme digestion and sequencing. Finally, the recombinant plasmid pIRES-neo-HBAg was transiently transfected to 293T cells by liposome method. The transfected 48h passed Western blot, flow cytometry and the transfection. The expression of pIRES-neo-HBAg was detected and analyzed by immunofluorescence. Then, the extracted recombinant plasmid was transfected into Hepa1-6 cells (BALB/c mouse hepatoma cells), and the optimum screening concentration of G418 was determined. The positive mixed clone was obtained after pressure screening, and the expression of HBV fusion antigen was detected by flow cytometry.
The results of the study:
The HBV new therapeutic replicator, DNA vaccine pSVK-HBVA, showed very strong cellular immunological activity when the immune dose was only 1 g. Under the specific antigen stimulus, the IFN- gamma factor of the lymphocyte was released significantly and the CD8+T cells were immunized. The CCK-8 detection showed that the spleen lymphocytes of the immunization group showed that the spleen lymphocytes were very good. The ELISA detection of cytokine release showed that the cell immune response activated by the vaccine was shifted to the Th1 reaction. In addition, the recombinant plasmid pIRES-neo-HBAg was successfully constructed by enzyme digestion and sequencing analysis. After transient transfection of 293T cells, the HBV fusion gene was expressed by Western blot detection, and the expression of the HBV fusion gene was expressed. The results of flow cytometry showed that the positive cell rate of the fusion antigen HBAg was 20.43%, and the results of immunofluorescence detection showed that the recombinant plasmid pIRES-neo-HBAg transfected cells showed red fluorescence and obtained effective expression. Then, the recombinant plasmid pIRES-neo-HBAg was transfected into Hepa1-6 cells and screened by 800 mu g/ml G418. A hybrid clone cell expressing stable HBV fusion antigen was obtained. Flow cytometry showed that the positive rate of HBV fusion antigen gene reached 86.20%.
The conclusions are as follows:
The new therapeutic replicon DNA vaccine pSVK-HBVA activates the efficient cellular immune response in the case of low immune dose, and the activated immune response is shifted to the Th1 type response. It is beneficial to improve the deficiency of conventional DNA immunogenicity and to overcome the immune tolerance of chronic hepatitis B. In addition, this study also constructed the expression of hepatitis B in this study. The eukaryotic expression plasmid pIRES-neo-HBAg of a variety of complex antigens has been preliminarily screened for the hybrid cloned cell lines, which lays the foundation for the establishment of an effective HBV cell model and a convenient animal model of tumor bearing mice.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392

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