免疫噬菌体抗体库的建立和人源TAT-Fab抗体的制备
发布时间：2018-10-31 16:21
【摘要】：目的: 破伤风是由破伤风杆菌侵入机体后引起的一种急性感染性疾病,主要是由破伤风杆菌产生的外毒素侵袭神经系统而导致的。目前唯一有效的防治手段是及时注射精制破伤风抗毒素(TAT)或人破伤风免疫球蛋白(TIG),以中和血液中游离的毒素。但由于TAT存在过敏反应,而TIG血液来源困难并存在病原微生物污染等问题,使临床应用上受到了很大的限制。近年来随着噬菌体抗体库技术的建立和发展,使人源抗体的制备成为可能,从而可以在根本上解决了过敏反应、血液不足及病原微生物污染等问题。本论文利用此免疫噬菌体抗体库技术,探索制备人抗破伤风类毒素的抗体。从破伤风类毒素免疫的个体获得人Fab抗体基因,构建人Fab抗体基因库;用破伤风外毒素筛选出展示人源TAT-Fab抗体噬菌体;挑选一株在大肠杆菌中表达可溶性人源TAT-Fab抗体,并初步鉴定所制备的可溶性人源TAT-Fab抗体具有部分中和破伤风外毒素的生物活性。 方法: 1.自破伤风类毒素加强免疫者的外周血中分离单个核细胞(PBMC),提取细胞总RNA并反转录合成cDNA;以cDNA为模板,用PCR法扩增全套人Ig轻链(κ链、λ链)基因和重链(γ链)Fd段基因。 2.自含有噬菌粒pComb3的大肠杆菌XL1-Blue中抽提并纯化载体pComb3。 3.XbaI和SacI双酶切混合轻链基因和噬菌粒pComb3,并从凝胶中回收纯化。T4 DNA连接酶连接上述双酶切产物,电穿孔转染大肠杆菌XL1-Blue,构建人Ig轻链基因库(pComb3-L)。XbaI和XhoI双酶切鉴定。 4.抽提纯化人Ig轻链基因库的重组噬菌粒(pComb3-L),与混合重链Fd段基因均XhoI和SpeI双酶切,并从凝胶中回收纯化。T4 DNA连接酶连接上述双酶切产物,电穿孔转染大肠杆菌XL1-Blue,构建人Fab抗体基因库(pComb3-HL)。XbaI和XhoI双酶切鉴定。
[Abstract]:Objective: tetanus is an acute infectious disease caused by tetanus. At present, the only effective prevention and treatment is timely injection of refined tetanus antitoxin (TAT) or human tetanus immunoglobulin (TIG), to neutralize the free toxin in blood. However, because of the allergic reaction of TAT, the difficulty of blood source of TIG and the contamination of pathogenic microorganism, the clinical application is restricted greatly. In recent years, with the establishment and development of phage antibody library technology, the preparation of human antibody becomes possible, which can fundamentally solve the problems of allergic reaction, blood shortage and pathogenic microorganism contamination. In this paper, the antibody against tetanus toxoid was prepared by using the technique of immune phage antibody library. Human Fab antibody gene was obtained from individuals immunized with tetanus toxoid, human Fab antibody gene library was constructed and human TAT-Fab antibody phage was screened by tetanus exotoxin. One strain was selected to express soluble human TAT-Fab antibody in Escherichia coli and the bioactivity of partially neutralizing tetanus exotoxin was preliminarily identified. Methods: 1. Isolation of mononuclear cell (PBMC), from peripheral blood of tetanus toxoid immunized individuals to extract total RNA and reverse synthesize cDNA; Using cDNA as template, a complete set of human Ig light chain (位 chain) and heavy chain (纬 chain) Fd segment genes were amplified by PCR. 2. Extraction and purification of vector pComb3. from Escherichia coli XL1-Blue containing bacteriophage pComb3 Light chain gene and phagocyte pComb3, were digested by 3.XbaI and SacI and purified from the gel. T4 DNA ligase was ligated into the double digested product and electroporation was transfected into Escherichia coli XL1-Blue,. The light chain gene bank of human Ig (pComb3-L). XbaI and XhoI) was constructed. 4. Recombinant macrophages (pComb3-L) from human Ig light chain gene bank were extracted and digested with both XhoI and SpeI of mixed heavy chain Fd gene, and purified from the gel. T4 DNA ligase was linked to the above double digested products. Human Fab antibody gene library was constructed by electroporation transfection of Escherichia coli XL1-Blue, (pComb3-HL). XbaI and XhoI).
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
本文编号：2302849
[Abstract]:Objective: tetanus is an acute infectious disease caused by tetanus. At present, the only effective prevention and treatment is timely injection of refined tetanus antitoxin (TAT) or human tetanus immunoglobulin (TIG), to neutralize the free toxin in blood. However, because of the allergic reaction of TAT, the difficulty of blood source of TIG and the contamination of pathogenic microorganism, the clinical application is restricted greatly. In recent years, with the establishment and development of phage antibody library technology, the preparation of human antibody becomes possible, which can fundamentally solve the problems of allergic reaction, blood shortage and pathogenic microorganism contamination. In this paper, the antibody against tetanus toxoid was prepared by using the technique of immune phage antibody library. Human Fab antibody gene was obtained from individuals immunized with tetanus toxoid, human Fab antibody gene library was constructed and human TAT-Fab antibody phage was screened by tetanus exotoxin. One strain was selected to express soluble human TAT-Fab antibody in Escherichia coli and the bioactivity of partially neutralizing tetanus exotoxin was preliminarily identified. Methods: 1. Isolation of mononuclear cell (PBMC), from peripheral blood of tetanus toxoid immunized individuals to extract total RNA and reverse synthesize cDNA; Using cDNA as template, a complete set of human Ig light chain (位 chain) and heavy chain (纬 chain) Fd segment genes were amplified by PCR. 2. Extraction and purification of vector pComb3. from Escherichia coli XL1-Blue containing bacteriophage pComb3 Light chain gene and phagocyte pComb3, were digested by 3.XbaI and SacI and purified from the gel. T4 DNA ligase was ligated into the double digested product and electroporation was transfected into Escherichia coli XL1-Blue,. The light chain gene bank of human Ig (pComb3-L). XbaI and XhoI) was constructed. 4. Recombinant macrophages (pComb3-L) from human Ig light chain gene bank were extracted and digested with both XhoI and SpeI of mixed heavy chain Fd gene, and purified from the gel. T4 DNA ligase was linked to the above double digested products. Human Fab antibody gene library was constructed by electroporation transfection of Escherichia coli XL1-Blue, (pComb3-HL). XbaI and XhoI).
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
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