慢性乙肝治疗性复制子DNA疫苗pSVK-HBVA细胞免疫活性研究和细胞模型的初步建立

发布时间：2018-06-17 09:25

本文选题：乙型肝炎病毒 + 复制子DNA疫苗　；参考：《第四军医大学》2013年硕士论文

【摘要】：研究背景：乙型肝炎病毒（Hepatitis B virus, HBV）慢性感染多年来已成为影响公共健康的世界性难题之一，每年大约有100万人死于HBV慢性感染所致的肝硬化、肝衰竭和肝细胞癌。迄今为止，HBV慢性感染尚无有效治疗手段，通过治疗性疫苗激发机体自身免疫是治疗慢性乙肝的重要途径。课题组在治疗性疫苗领域多年研究探索，自主研发的新型复制子DNA疫苗载体系统pSVK，能在常规DNA疫苗1/100的剂量即可激发高效的细胞免疫应答。针对慢性乙肝治疗中的核心难题“免疫耐受”，实验室前期构建了融合多种免疫增效策略的新型复制子DNA疫苗pSVK-HBVA，使其在靶抗原摄入、表达、呈递和免疫应答激活等多个方面具备突出优势，有利于打破慢性乙肝的免疫耐受。本研究拟开展乙肝新型治疗性疫苗pSVK-HBVA免疫小鼠后的细胞免疫应答研究，同时为检测新型疫苗pSVK-HBVA的细胞毒T淋巴细胞（cytotoxicity Tlymphocyte,CTL）活性，还构建了融合HBV多种抗原基因的重组表达质粒pIRES-neo-HBAg。研究目的：开展新型复制子DNA疫苗pSVK-HBVA免疫小鼠后的细胞免疫应答研究，进一步验证经过疫苗优化设计后的新型治疗性复制子DNA疫苗的免疫治疗功效。此外，还构建了包含HBV多个抗原区段的真核表达质粒pIRES-neo-HBAg，初步筛选了混合克隆细胞株，为进一步筛选稳定表达HBV融合抗原的单克隆细胞株奠定实验基础。研究方法：首先，以本研究室前期构建的HBV新型治疗性复制子DNA疫苗pSVK-HBVA免疫BALB/c小鼠，末次免疫后一周进行分离小鼠脾脏淋巴细胞，通过IFN-γ释放的酶联免疫斑点（ELISPOT）法检测免疫后小鼠的CD8+T细胞活性，CCK-8（Cell Counting Kit-8）法检测淋巴细胞在特异性乙肝抗原刺激下的免疫增殖活性，ELISA法检测脾淋巴细胞免疫相关细胞因子包括Th1类因子IL-2、IFN-γ和Th2类因子IL-4、IL-10的免疫释放；其次，为进一步研究该疫苗激活的CTL免疫反应，以pVAX1-HBVA为模板，设计引物进行PCR扩增包含HBV前S1、前S2、HBsAg和HBcAg的融合抗原基因（命名为HBAg），克隆入真核表达载体pIRES-neo中，构建重组表达质粒pIRES-neo-HBAg，并进行酶切和测序验证；最后将重组质粒pIRES-neo-HBAg进行脂质体法瞬时转染293T细胞，转染后48h通过Western blot、流式细胞术及免疫荧光等方法检测和分析其表达情况，然后,再将提取的pIRES-neo-HBAg重组质粒转染Hepa1-6细胞（BALB/c小鼠来源的肝癌细胞），确定G418的最佳筛选浓度，加压筛选后获得阳性混合克隆，流式细胞术检测HBV融合抗原的表达。研究结果： HBV新型治疗性复制子DNA疫苗pSVK-HBVA，在免疫剂量仅为1μg时就表现出很强的细胞免疫学活性。在特异性抗原刺激下疫苗免疫组小鼠淋巴细胞IFN-γ因子释放显著，CD8+T细胞被免疫激活；CCK-8检测显示疫苗免疫组小鼠的脾淋巴细胞显示出很强的增殖活性；细胞因子释放的ELISA检测显示，疫苗免疫后激活的细胞免疫反应向Th1类反应偏移。此外，重组质粒pIRES-neo-HBAg经酶切鉴定和测序分析，证实构建成功；瞬时转染293T细胞后，经Western blot检测显示， HBV融合基因得到表达，，且分子量大小正确；流式细胞术检测显示，表达融合抗原HBAg的阳性细胞率为20.43%；免疫荧光检测结果显示重组质粒pIRES-neo-HBAg转染细胞显示红色荧光，获得有效表达。随后，将重组质粒pIRES-neo-HBAg转染Hepa1-6细胞，并用800μg/ml的G418加压筛选，初步筛选获得了稳定表达HBV融合抗原的混合克隆细胞，流式细胞术检测显示HBV融合抗原基因的阳性细胞率可达86.20%。研究结论：新型治疗性复制子DNA疫苗pSVK-HBVA在免疫剂量较低的情况下即可激活高效的细胞免疫应答，且激活的免疫反应向Th1类应答偏移，有利于改善常规DNA免疫原性不足的问题，也有利于克服慢性乙肝的免疫耐受。此外，本研究还构建了表达乙肝多种复合抗原的真核表达质粒pIRES-neo-HBAg，初步筛选了混合克隆细胞株，为建立有效的HBV体外细胞模型和方便的荷瘤小鼠动物模型奠定了基础。
[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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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