小鼠LIGHT真核表达载体的构建及其与乙肝核酸疫苗的联合免疫研究

发布时间：2018-08-26 10:07

【摘要】： 感染HBV能导致急性、慢性肝炎,与肝硬化及肝细胞癌有密切关系。目前,控制HBV感染的有效方法是接种乙肝疫苗。基因疫苗的兴起和发展为乙型肝炎病毒感染的防治提供了新的途径。基因疫苗可诱导机体产生细胞及体液免疫反应,特别是诱导细胞免疫反应的能力优于蛋白、多肽类疫苗,在乙型肝炎病毒的清除方面具有较好的应用前景,更适应于慢性病毒感染的预防与治疗。核酸疫苗的免疫效果在不同动物个体中差异较大,某些肿瘤及病毒的T、B细胞表位抗原性弱,难以诱导有效的T、B细胞免疫应答,提高核酸疫苗的免疫效果已成为研究热点。基因佐剂是将编码某些细胞因子及T、B细胞表面共刺激分子等的基因与目的基因克隆于同一载体或不同载体共同免疫动物,其表达的相应蛋白可起到佐剂的作用,从而大大增强T、B细胞的免疫应答水平。 LIGHT是TNF配体家族的成员,可以共刺激T细胞增殖。HVEM是LIGHT介导T细胞活化的受体,LIGHT通过与HVEM结合发挥共刺激作用,参与T细胞增殖和细胞因子的产生。越来越多的证据表明LIGHT与其受体的相互作用可作为控制细胞免疫应答的潜在目标。本研究对小鼠LIGHT作为乙肝核酸疫苗的基因佐剂方面进行了初步研究。在克隆小鼠LIGHT基因的基础上,构建其胞外段基因的原核表达载体,在大肠杆菌中进行诱导表达并对其表达条件进行了初步的探索;构建LIGHT全长基因的真核表达质粒并在体外转染细胞,检测结果表明真核表达质粒中的LIGHT基因可在哺乳动物细胞中进行表达;混合淋巴细胞反应中LIGHT转染的树突状细胞可显著增强同种异体T淋巴细胞增殖;将LIGHT真核表达质粒与乙肝核酸疫苗质粒联合免疫Balb/c小鼠,细胞免疫与体液免疫功能检测结果表明LIGHT可以增强小鼠体内的特异性免疫应答,从而发挥免疫佐剂的作用。
[Abstract]:Infection with HBV can lead to acute and chronic hepatitis, closely related to cirrhosis and hepatocellular carcinoma. At present, the effective way to control HBV infection is to inoculate hepatitis B vaccine. The rise and development of gene vaccine provide a new way for the prevention and treatment of hepatitis B virus infection. Gene vaccine can induce cellular and humoral immune response, especially the ability of inducing cellular immune response is superior to that of protein and polypeptide vaccine. More suitable for the prevention and treatment of chronic virus infection. The immune effect of nucleic acid vaccine varies greatly among different animals. The antigenicity of TnB cell epitopes of some tumors and viruses is weak, it is difficult to induce an effective TnB cell immune response, and improving the immune effect of nucleic acid vaccine has become a hot research topic. Gene adjuvant is a kind of gene which encodes some cytokines and costimulatory molecules on the surface of TnB cells and is cloned into the same vector or different vectors to immunize animals. The corresponding protein expressed by the adjuvant can act as an adjuvant. LIGHT is a member of TNF ligand family, which can co-stimulate T cell proliferation. HVEM is the receptor of T cell activation mediated by LIGHT. Participate in T cell proliferation and cytokine production. There is growing evidence that the interaction of LIGHT with its receptors may be a potential target for controlling cellular immune responses. In this study, we studied mouse LIGHT as gene adjuvant of hepatitis B nucleic acid vaccine. On the basis of cloning mouse LIGHT gene, the prokaryotic expression vector of its extracellular segment gene was constructed, and the expression conditions were preliminarily explored in Escherichia coli. The eukaryotic expression plasmid of the full-length LIGHT gene was constructed and transfected into the cells in vitro. The results showed that the LIGHT gene in the eukaryotic expression plasmid could be expressed in mammalian cells. In mixed lymphocyte reaction, dendritic cells transfected with LIGHT could significantly enhance the proliferation of allogeneic T lymphocytes, and Balb/c mice were immunized with LIGHT eukaryotic expression plasmid and hepatitis B nucleic acid vaccine plasmid. The results of cellular and humoral immunity showed that LIGHT could enhance the specific immune response in mice and play the role of immune adjuvant.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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