【摘要】：目的: 破傷風(fēng)是由破傷風(fēng)桿菌侵入機(jī)體后引起的一種急性感染性疾病,主要是由破傷風(fēng)桿菌產(chǎn)生的外毒素侵襲神經(jīng)系統(tǒng)而導(dǎo)致的。目前唯一有效的防治手段是及時(shí)注射精制破傷風(fēng)抗毒素(TAT)或人破傷風(fēng)免疫球蛋白(TIG),以中和血液中游離的毒素。但由于TAT存在過敏反應(yīng),而TIG血液來源困難并存在病原微生物污染等問題,使臨床應(yīng)用上受到了很大的限制。近年來隨著噬菌體抗體庫(kù)技術(shù)的建立和發(fā)展,使人源抗體的制備成為可能,從而可以在根本上解決了過敏反應(yīng)、血液不足及病原微生物污染等問題。本論文利用此免疫噬菌體抗體庫(kù)技術(shù),探索制備人抗破傷風(fēng)類毒素的抗體。從破傷風(fēng)類毒素免疫的個(gè)體獲得人Fab抗體基因,構(gòu)建人Fab抗體基因庫(kù);用破傷風(fēng)外毒素篩選出展示人源TAT-Fab抗體噬菌體;挑選一株在大腸桿菌中表達(dá)可溶性人源TAT-Fab抗體,并初步鑒定所制備的可溶性人源TAT-Fab抗體具有部分中和破傷風(fēng)外毒素的生物活性。 方法: 1.自破傷風(fēng)類毒素加強(qiáng)免疫者的外周血中分離單個(gè)核細(xì)胞(PBMC),提取細(xì)胞總RNA并反轉(zhuǎn)錄合成cDNA;以cDNA為模板,用PCR法擴(kuò)增全套人Ig輕鏈(κ鏈、λ鏈)基因和重鏈(γ鏈)Fd段基因。 2.自含有噬菌粒pComb3的大腸桿菌XL1-Blue中抽提并純化載體pComb3。 3.XbaI和SacI雙酶切混合輕鏈基因和噬菌粒pComb3,并從凝膠中回收純化。T4 DNA連接酶連接上述雙酶切產(chǎn)物,電穿孔轉(zhuǎn)染大腸桿菌XL1-Blue,構(gòu)建人Ig輕鏈基因庫(kù)(pComb3-L)。XbaI和XhoI雙酶切鑒定。 4.抽提純化人Ig輕鏈基因庫(kù)的重組噬菌粒(pComb3-L),與混合重鏈Fd段基因均XhoI和SpeI雙酶切,并從凝膠中回收純化。T4 DNA連接酶連接上述雙酶切產(chǎn)物,電穿孔轉(zhuǎn)染大腸桿菌XL1-Blue,構(gòu)建人Fab抗體基因庫(kù)(pComb3-HL)。XbaI和XhoI雙酶切鑒定。
[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 (緯 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).
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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本文編號(hào)：2302849