肝脏F蛋白的表达及单克隆抗体的制备

发布时间：2017-12-31 20:41

本文关键词：肝脏F蛋白的表达及单克隆抗体的制备　出处：《天津医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 肝脏疾病严重影响人类健康,并对社会经济发展产生巨大影响。肝癌是世界上死亡率最高的恶性肿瘤之一,病毒性肝炎、肝纤维化、肝硬化等都是肝癌主要的致病原。因此肝病的诊断就成为医学诊断学的重要组成部分。人类肝脏F蛋白是一种新型的尚未应用于临床的直接反映肝损伤程度的生物学标志之一。从80年代开始,人们开始研究血清F蛋白含量与肝损伤程度的关系,获得了一些临床数据。肝细胞中的F蛋白浓度与血清F蛋白的浓度之间存在的巨大差异以及F蛋白的严格的组织特异性,使其成为一种非常有希望的反映肝细胞损伤程度的灵敏和特异的指标。目前,肝脏特异性F蛋白的研究与临床应用受到越来越多的关注。本文在前人研究的基础上,将F蛋白的表达质粒pET15b-F转化到表达菌株BL21(DE3)pLysS中,用IPTG诱导其表达,表达后通过亲和层析柱(Ni~(2+)-chargedIDA His-bind column)纯化,用十二烷基硫酸钠-聚丙稀酰胺凝胶电泳与免疫印迹(Western blot)对其进行检测。并以此蛋白为抗原免疫BALB-C小鼠以获得免疫的脾细胞,用间接ELISA法测定其血清抗体的效价,选取抗体效价高的小鼠取脾细胞与骨髓瘤细胞融合,制备单克隆抗体。人F蛋白的表达菌株BL21(DE3)pLysS表达后的全菌蛋白进行SDS-PAGE电泳检测,目的条带分子量大小约为43kD,与文献报导一致。Western-blot显示纯化后的F蛋白与豚鼠抗人提纯F蛋白多克隆抗血清反应为阳性,说明我们经基因工程方法得到的纯化的F蛋白具有免疫活性。免疫BALB-C小鼠后,用ELISA法测定血清其抗体滴度高达1∶72000,可用于单抗的制备;细胞融合后培养上清有15孔抗体阳性,抗体滴度最高为1:25600,Western-blot显示制备的抗体与基因工程表达的人肝脏特异性F蛋白发生了反应,表明我们制备的抗体具有免疫特异性,可用于后续的单克隆抗体的大量制备和提纯以及免疫试剂的开发。肝脏特异性F蛋白是反映肝损伤的一种灵敏、有效而又稳定的指标,同时F蛋白在肝病早期诊断与治疗、药物的筛选与疗效观察、治愈状况、肝损伤程度的判断等方面以及法医学方面、免疫耐受肝移植抗排斥方面的研究都具有重要的应用价值。用化学提纯的方法很难得到大量F蛋白,这就阻碍了其他研究的进行。因此,本文采用基因工程方法得到人肝脏F蛋白的表达克隆,并诱导表达人肝脏F蛋白,解决了其来源问题。同时以此基因工程蛋白为抗原初步制备单克隆抗体,并通过免疫印迹法证实了此抗体的特异性,为下一步的免疫试剂最适条件的摸索、免疫耐受及其免疫学特性等研究打下基础,为临床肝病的诊断和治疗打开新的一扇门。
[Abstract]:Liver diseases seriously affect human health, and have a huge impact on the social and economic development. Liver cancer is one of malignant tumors with the highest mortality rate in the world, viral hepatitis, liver fibrosis, liver cirrhosis and hepatocellular carcinoma are the main pathogens. Therefore the diagnosis of liver disease has become an important part of medical diagnostics. The F protein in human liver is a new kind of biological mark is not applied to clinical directly reflect the degree of liver injury. From the beginning of 80s, people began to study the relationship between serum F protein content and the degree of liver injury, get some clinical data. There is a huge difference between the concentration of F protein in liver cells and the serum concentration of F protein and F protein the strict tissue specificity, make it become a very promising to reflect the degree of liver cell injury of the sensitive and specific index. At present, the research of the liver specific F protein More and more attention has been paid to the clinical application.
In this paper, on the basis of previous studies, the expression of transforming plasmid pET15b-F F protein expression strain BL21 (DE3) pLysS, induced the expression of IPTG expression by affinity chromatography (Ni~ (2+) -chargedIDA His-bind column) with twelve purification, sodium dodecyl sulfate - poly acrylamide gel electrophoresis and Western blot (Western blot) was used to detect the protein. Then BALB-C mice were immunized with immune spleen cells, the serum antibody titer was determined by indirect ELISA method, selection of high antibody titer of mice spleen cells and myeloma cell fusion, preparation of monoclonal antibody.
Human F protein expression strain BL21 (DE3) and the protein expression of pLysS was detected by SDS-PAGE, the target band size of molecular weight is about 43kD, and the purified F protein and F protein purified guinea pig anti human polyclonal antibody response was positive reported consistent.Western-blot showed that we by gene engineering method the purified F protein with immunological activity. After immunization of BALB-C mice, the ELISA method for the determination of serum antibody titers were as high as 1 to 72000, can be used for the preparation of the monoclonal antibody; cell culture supernatant after fusion of 15 hole antibody positive and antibody titer as high as 1:25600, Western-blot shows the reaction expression of antibody and gene engineering the preparation of human liver specific F protein showed that the antibodies we prepared has immune specificity, can be used for the large number of monoclonal antibody preparation and purification and immunological reagents.
The liver specific F protein is a reflection of liver injury in a sensitive, effective and stable index, and F protein in the early diagnosis and treatment of liver disease, screening and efficacy of drugs, cure condition, the degree of liver injury and forensic judgment, has very important application value of anti immune tolerance in liver transplantation rejection. With chemical purification method and it is very difficult to get a large number of F proteins, which hampered the other research. Therefore, cloning and expression of F protein in human liver by using gene engineering method, and induce the expression of F protein in human liver, to solve the problem. At the same time as source of genetic engineering protein for preliminary preparation monoclonal antibody and antigen specificity of this antibody was confirmed by immunoblotting, ELISA is the most suitable condition of next exploration, to lay the foundation for the study of immune tolerance and its immunological characteristics, Open a new door for the diagnosis and treatment of clinical liver disease.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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