土拨鼠CTLA4的原核表达和多克隆抗体的制备

发布时间：2018-01-06 12:06

本文关键词：土拨鼠CTLA4的原核表达和多克隆抗体的制备　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：研究背景: 土拨鼠肝炎病毒(Woodchuck hepatitis virus,WHV)属于嗜肝DNA病毒,与人乙型肝炎病毒(Hepatitis B virus,HBV)关系十分密切,在基因组结构、病毒复制和病毒感染过程等方面都十分的相类似[1,2,3],是目前已知的用于研究乙肝病毒的致病机制和筛选新的抗病毒药物,并且在研制新的疫苗中发挥重要作用的最理想小动物模型。目前各国学者对乙型肝炎病毒难以清除,如果机体缺乏有效的T细胞免疫会最终导致慢性化这一点是持一致和肯定意见的。注重于探讨抗病毒反应引起T细胞活化的相关因素在以往的研究中多有涉及,但是却忽略了一个目前已经逐渐认识到了的重要问题:负调节机制在抑制T细胞活化方面同样发挥重要作用。近年来的许多研究表明乙肝感染程度除了与免疫反应的强度有关之外,它同时又是一个被许多细胞因子相互调节的过程,这其中就包括细胞表面的受体CTLA4[4]。在本研究中,通过分子克隆的手段成功将CTLA4构建到原核表达载体中并在大肠杆菌BL21(DE3)进行诱导表达,并最终制备了多克隆抗体。这一实验结果为进一步完善土拨鼠模型相关指标,检测和研究CTLA4在正常、急性、慢性WHV中的表达,评估CTLA4在肝炎慢性化中的作用奠定一定的基础。目的 1.构建土拨鼠CTLA4的原核表达载体并在大肠杆菌中进行表达。 2.制备了多克隆抗体,同时检测土拨鼠CTLA4的特异性和灵敏性。方法 1.应用基因工程技术将土拨鼠的CTLA4的基因片段装入原核表达载体,转化表达菌后诱导目的蛋白的表达,应用电洗脱方法纯化目的蛋白,并用SDS-PAGE和Western blot对表达产物(目的蛋白)进行相应鉴定。 2.用纯化的目的蛋白免疫新西兰大白兔制备多克隆抗体,并纯化多克隆抗体。结果 1.通过PCR鉴定、酶切鉴定,并在核酸测序后与CTLA4的原始序列比对,结果表明成功构建土拨鼠CTLA4的原核表达载体。 2.用目的蛋白免疫新西兰大白兔获得多克隆抗体,采用酶联免疫吸附实验(ELISA),Western blot等方法对多克隆抗体的灵敏度和特异性进行检测。
[Abstract]:Research background:
Woodchuck hepatitis virus (Woodchuck hepatitis, virus, WHV) belongs to the hepatotropic DNA virus, and hepatitis B virus (Hepatitis B, virus, HBV) very close relationship, in the genomic structure, viral replication and virus infection and other aspects are very similar to [1,2,3], is currently known for the pathogenic mechanism of hepatitis B virus and screening of new antiviral drugs, the animal model and play an important role in the development of new vaccines. At present, many scholars are difficult to remove of the hepatitis B virus, if the body lacks the effective immune T cells will eventually lead to the chronicity of this point is consistent and positive opinion. Focus on the study of the antiviral response caused by there are factors involved in the activation of T cells than in previous studies, but it ignores an important problem which has been gradually aware of the negative regulation mechanism in the inhibition of T cells Also play an important role in activation. In recent years many studies show that the degree of hepatitis B infection in addition to the intensity of the immune reaction, it is also a lot of cytokines regulate each other processes, including the cell surface receptor CTLA4[4].
In this study, by means of molecular cloning CTLA4 successfully constructed into the prokaryotic expression vector in Escherichia coli BL21 (DE3) induced expression, and finally prepared the polyclonal antibody. The results of this experiment is to further improve the relevant index marmot rat model, detection and research of CTLA4 in normal, acute. The expression of WHV in chronic, laid the foundation for evaluation of CTLA4 in the chronic hepatitis.
objective
1. to construct the prokaryotic expression vector of woodchuck CTLA4 and expressed in Escherichia coli.
2. preparation of polyclonal antibody, simultaneous detection of woodchuck CTLA4 specificity and sensitivity.
Method
1. application of gene engineering technique of Marmot CTLA4 gene fragment into the prokaryotic expression vector for protein expression coli after the application of electrodialysis method to purify the protein, and use SDS-PAGE and Western to blot expression (protein) were identified.
2. purified to immunize New Zealand white rabbits to prepare polyclonal antibody, and purified polyclonal antibody.
Result
1. identified by PCR, enzyme digestion, and the original sequence alignment and CTLA4 in nucleic acid sequencing, the results indicated the successful construction of prokaryotic expression vector of woodchuck CTLA4.
2. to immunize rabbits to obtain polyclonal antibodies by enzyme-linked immunosorbent assay (ELISA) detection, the sensitivity and specificity of Western blot method of polyclonal antibody.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

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