抗剛地弓形蟲特異性人源抗體Fab片段的制備和研究
發(fā)布時間：2019-05-01 06:58
【摘要】：剛地弓形蟲(Toxoplasma gondii)簡稱弓形蟲,屬于頂復門、真球蟲目、弓形蟲科,是一種世界性分布的專性細胞內(nèi)寄生的機會性致病原蟲,可寄生于多種脊椎動物的體內(nèi)。免疫缺損者感染弓形蟲后,可導致嚴重的全身性弓形蟲病。可通過母體垂直感染胎兒出現(xiàn)先天性弓形蟲病。大量的實驗證明在弓形蟲感染過程中,機體產(chǎn)生的抗體具有重要的保護性作用。為此本研究旨在制備出抗弓形蟲的人源抗體,為弓形蟲病的被動免疫防治策略的發(fā)展和應用提供理論和實驗依據(jù)。 首先利用基因重組技術(shù)構(gòu)建弓形蟲速殖子表面蛋白SAG1的表達質(zhì)粒,通過原核表達系統(tǒng)進行大量的表達,并純化制備出具有生物活性的重組SAG1,作為抗體庫篩選抗原。分離弓形蟲病患者的外周血淋巴細胞,利用工程抗體技術(shù)構(gòu)建出庫容為3×10的人抗弓形蟲免疫球蛋白G基因文庫。采用克隆印跡法、ELISA、間接免疫熒光反應(IFA)等多種方法以重組蛋白對抗體庫進行較大規(guī)模的篩選驗證,對所得陽性克隆進行測序和結(jié)構(gòu)分析。經(jīng)過6×105個克隆的篩選,最后得到兩個陽性克隆,分別命名為Tox08、Toxll。Tox08和Toxll的重鏈可變區(qū)近似系同為VH3-23,基因同源性分別為98%和98.26%。Tox08的輕鏈可變區(qū)近似系為VK1-33、基因同源性為87.46%；Toxll的輕鏈可變區(qū)近似系為Vκ1-27、基因同源性為93.43%。根據(jù)Kabat系統(tǒng)對可變區(qū)的劃分,Toxll的輕鏈可變區(qū)部分缺少互補決定區(qū)1(CDR1),完全缺失CDR2和框架區(qū)FR2。為了提高抗體的親和力,分別對Tox08的輕重鏈和Toxll的輕鏈進行鏈替換,構(gòu)建鏈替換庫。經(jīng)篩選后,從Toxll的重鏈與弓形蟲病患者來源的輕鏈庫構(gòu)建的Toxll輕鏈替換庫ToxllH-Toxlib中篩選出兩個陽性克隆,分別命名為Tox87L-11H和Tox1403L-11H。Tox87L-11H和Tox1403L-11H的輕鏈可變區(qū)近似系同為VK：I-17,基因同源性分別為92.11%和89.61%。對陽性克隆Tox1403L-11H進行大量表達,并分別采用抗體親和層析、鈷離子親和層析和鎳離子親和層析三種純化方法對其進行純化。根據(jù)純化后的Fab片段的產(chǎn)量、輕重鏈比例以及與rSAG1的親和力評定純化方法的優(yōu)劣。經(jīng)抗體親和層析和鎳離子親和層析得到的Fab片段的輕重鏈比例為1：1,產(chǎn)量分別為0.75mg/L和1.08mg/L。鈷離子親和層析純化的Fab片段產(chǎn)量雖然高于另兩種方法,為1.16mg/L,但輕重鏈的比例為1：2。Biacore檢測分析表明,鎳離子親和層析提純的Fab片段與rSAG1的親和力最高,其結(jié)合常數(shù)為9.0×1071/M,解離常數(shù)為2.01×10-8M。 上述研究工作不僅制備出具有抗弓形蟲特異性的人源抗體Fab片段,還同時確立了其純化方法,為抗弓形蟲特異性的人源抗體工廠化生產(chǎn)和臨床研究應用提供了理論和實驗依據(jù)。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) (Toxoplasma gondii) belongs to Toxoplasma gondii (Toxoplasma gondii). Toxoplasma gondii (Toxoplasma gondii) is a worldwide distribution of opportunistic pathogenic protozoa, which can be found in many vertebrates. Infection with Toxoplasma gondii can lead to severe systemic toxoplasmosis. Congenital toxoplasmosis can occur through maternal vertical infection of the fetus. A large number of experiments have proved that the antibody produced by the organism plays an important protective role in the process of Toxoplasma gondii infection. The aim of this study is to prepare human antibodies against Toxoplasma gondii, and to provide theoretical and experimental basis for the development and application of passive immunocontrol strategies for toxoplasmosis. The expression plasmid of Toxoplasma Tachyzoite surface protein (SAG1) was constructed by gene recombination technique. The recombinant Toxoplasma gondii Toxoplasma Tachyzoite surface protein (SAG1,) was expressed by prokaryotic expression system. Peripheral blood lymphocytes from patients with toxoplasmosis were isolated and a human anti-Toxoplasma gondii immunoglobulin G gene library with 3 脳 10 reservoir capacity was constructed by engineering antibody technique. Clone blot method, ELISA, indirect immunofluorescence reaction (IFA) and other methods were used to screen and verify the recombinant protein antibody library on a large scale, and the positive clones were sequenced and analyzed. After screening 6 脳 10 5 clones, two positive clones were obtained, which were named Tox08,Toxll.Tox08 and Toxll, respectively. The approximate regions of the heavy chain variable region were the same as VH3-23,. The homology of the VK1-33, gene was 87.46% between 98% of the gene homology and 87.46% of the similarity of the light chain variable region of 98.26%.Tox08. The variable region of the light chain of Toxll was similar to V 魏 1, and the homology of the gene was 93.43%. According to the division of variable regions in Kabat system, there is a lack of complementary determinant region 1 (CDR1) in the light chain variable region of Toxll, and a complete absence of CDR2 and frame region FR2.. In order to improve the affinity of antibody, the light chain of Tox08 and the light chain of Toxll were replaced by chain, and the chain substitution library was constructed. After screening, two positive clones were screened from the Toxll light chain replacement library ToxllH-Toxlib constructed from the heavy chain library of Toxll and the light chain library from patients with toxoplasmosis. The similarity of the variable region of light chain named Tox87L-11H, Tox1403L-11H.Tox87L-11H and Tox1403L-11H was 92.11% and 89.61%, respectively. The homology of the VK:I-17, gene was 92.11% and 89.61%, respectively. The positive clone Tox1403L-11H was expressed in large quantities, and purified by antibody affinity chromatography, cobalt ion affinity chromatography and nickel ion affinity chromatography respectively. The purification method was evaluated according to the yield of the purified Fab fragment, the proportion of light and heavy chains and the affinity to rSAG1. The weight chain ratio of the Fab fragment obtained by antibody affinity chromatography and nickel ion affinity chromatography was 1 渭 1, and the yield was 1.08 mg 路L-1 and 1.08 mg 路L-1 0.75mg/L, respectively. Although the yield of Fab fragment purified by cobalt ion affinity chromatography was higher than the other two methods, it was 1.16mg / L, but the ratio of light and heavy chain was 1:2.Biacore. The results showed that the Fab fragment purified by nickel ion affinity chromatography had the highest affinity to rSAG1. The binding constant is 9.0 脳 1071 mm and the dissociation constant is 2.01 脳 10-8 M. The above research work not only prepared the Fab fragment of anti-Toxoplasma gondii-specific human antibody, but also established the purification method, which provided the theoretical and experimental basis for the industrial production and clinical application of anti-Toxoplasma gondii-specific human antibody.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392
本文編號：2469097
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) (Toxoplasma gondii) belongs to Toxoplasma gondii (Toxoplasma gondii). Toxoplasma gondii (Toxoplasma gondii) is a worldwide distribution of opportunistic pathogenic protozoa, which can be found in many vertebrates. Infection with Toxoplasma gondii can lead to severe systemic toxoplasmosis. Congenital toxoplasmosis can occur through maternal vertical infection of the fetus. A large number of experiments have proved that the antibody produced by the organism plays an important protective role in the process of Toxoplasma gondii infection. The aim of this study is to prepare human antibodies against Toxoplasma gondii, and to provide theoretical and experimental basis for the development and application of passive immunocontrol strategies for toxoplasmosis. The expression plasmid of Toxoplasma Tachyzoite surface protein (SAG1) was constructed by gene recombination technique. The recombinant Toxoplasma gondii Toxoplasma Tachyzoite surface protein (SAG1,) was expressed by prokaryotic expression system. Peripheral blood lymphocytes from patients with toxoplasmosis were isolated and a human anti-Toxoplasma gondii immunoglobulin G gene library with 3 脳 10 reservoir capacity was constructed by engineering antibody technique. Clone blot method, ELISA, indirect immunofluorescence reaction (IFA) and other methods were used to screen and verify the recombinant protein antibody library on a large scale, and the positive clones were sequenced and analyzed. After screening 6 脳 10 5 clones, two positive clones were obtained, which were named Tox08,Toxll.Tox08 and Toxll, respectively. The approximate regions of the heavy chain variable region were the same as VH3-23,. The homology of the VK1-33, gene was 87.46% between 98% of the gene homology and 87.46% of the similarity of the light chain variable region of 98.26%.Tox08. The variable region of the light chain of Toxll was similar to V 魏 1, and the homology of the gene was 93.43%. According to the division of variable regions in Kabat system, there is a lack of complementary determinant region 1 (CDR1) in the light chain variable region of Toxll, and a complete absence of CDR2 and frame region FR2.. In order to improve the affinity of antibody, the light chain of Tox08 and the light chain of Toxll were replaced by chain, and the chain substitution library was constructed. After screening, two positive clones were screened from the Toxll light chain replacement library ToxllH-Toxlib constructed from the heavy chain library of Toxll and the light chain library from patients with toxoplasmosis. The similarity of the variable region of light chain named Tox87L-11H, Tox1403L-11H.Tox87L-11H and Tox1403L-11H was 92.11% and 89.61%, respectively. The homology of the VK:I-17, gene was 92.11% and 89.61%, respectively. The positive clone Tox1403L-11H was expressed in large quantities, and purified by antibody affinity chromatography, cobalt ion affinity chromatography and nickel ion affinity chromatography respectively. The purification method was evaluated according to the yield of the purified Fab fragment, the proportion of light and heavy chains and the affinity to rSAG1. The weight chain ratio of the Fab fragment obtained by antibody affinity chromatography and nickel ion affinity chromatography was 1 渭 1, and the yield was 1.08 mg 路L-1 and 1.08 mg 路L-1 0.75mg/L, respectively. Although the yield of Fab fragment purified by cobalt ion affinity chromatography was higher than the other two methods, it was 1.16mg / L, but the ratio of light and heavy chain was 1:2.Biacore. The results showed that the Fab fragment purified by nickel ion affinity chromatography had the highest affinity to rSAG1. The binding constant is 9.0 脳 1071 mm and the dissociation constant is 2.01 脳 10-8 M. The above research work not only prepared the Fab fragment of anti-Toxoplasma gondii-specific human antibody, but also established the purification method, which provided the theoretical and experimental basis for the industrial production and clinical application of anti-Toxoplasma gondii-specific human antibody.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前1條
1 李越希;陶開華;張錦海;潘明潔;;弓形蟲P30蛋白抗原表位的克隆表達及純化鑒定[J];中國生物制品學雜志;2006年03期
，本文編號：2469097
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