【作者】 张亚丽；

【导师】 江文正；

【作者基本信息】 华东师范大学 ， 生物医学， 2008， 硕士

【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。更多还原

【Abstract】 Infection of HBV can lead to acute or chronic hepatitis and it relates closely to cirrhosis and hepatocellular carcinoma.An effective method to control the HBV infection is administration of HBV vaccine.The rising and development of DNA vaccine provide a new way for prevention and treatment of HBV infection.Genetic vaccines could induce protective immune responses in several animal models,it is superior to recombinant protein vaccine and peptide vaccine,especially in inducing cellular immune responses.DNA vaccine,which has a good application prospect in elimination of HBV,is more suitable for prevention and therapy of chronic virus infections.There is a great difference among the immune effects of DNA vaccine in different animals.It is difficult for some tumor and virus antigens with weak T or B cell epitope to induce efficient responses and many strategies have been employed to improve the effect of nucleic acid vaccines.Genetic adjuvant is constructed by cloning genes encode cytokines and costimulators into the same vector or different vectors with target genes.After coimmunization with DNA vaccine,the expressed protein could enhance the immune responses.LIGHT is a member of the TNF superfamily and functions as a costimulatory molecule for T cell proliferation.LIGHT plays a role in costimulating and takes part in T cell proliferation as well as the production of cytokines through combining with HVEM,a receptor of LIGHT for mediating T cell activation.More and more researches suggest that the interaction of LIGHT and its receptors could be a potential target for controlling cellular immune responses.In this research,the LIGHT gene was cloned and the adjuvant effect of LIGHT for HBV DNA vaccine was studied in a murine model.The extracellular domain of LIGHT was cloned into a prokaryotic expression plasmid and the expression conditions of recombinant protein were studied.The eukaryotic expression plasmid was constructed by inserting full-length LIGHT gene into pcDNA3.1(+)and transfection in vitro has demonstrated that foreign protein was expressed successfully. MLR assay showed that DCs transfected by LIGHT eukaryotic expression plasmid induced markedly higher allogeneic lymphocyte proliferation than DCs transfected by pcDNA3.1(+)vector plasmid and untreated DCs at all dilutions.Balb/c mice were immunized with HBV DNA vaccine plasmids alone or in combination with LIGHT expression plasmids.Detection of humoral and cellular immune responses suggested that LIGHT expression plasmids can elicit stronger specific immune responses in mice than HBV DNA vaccine plasmids alone,and LIGHT may be an effective immunological adjuvant in HBV DNA vaccination.更多还原