抗剛地弓形蟲(chóng)特異性人源抗體Fab片段的制備和研究
發(fā)布時(shí)間：2019-05-01 06:58
【摘要】：剛地弓形蟲(chóng)(Toxoplasma gondii)簡(jiǎn)稱弓形蟲(chóng),屬于頂復(fù)門(mén)、真球蟲(chóng)目、弓形蟲(chóng)科,是一種世界性分布的專性細(xì)胞內(nèi)寄生的機(jī)會(huì)性致病原蟲(chóng),可寄生于多種脊椎動(dòng)物的體內(nèi)。免疫缺損者感染弓形蟲(chóng)后,可導(dǎo)致嚴(yán)重的全身性弓形蟲(chóng)病�？赏ㄟ^(guò)母體垂直感染胎兒出現(xiàn)先天性弓形蟲(chóng)病。大量的實(shí)驗(yàn)證明在弓形蟲(chóng)感染過(guò)程中,機(jī)體產(chǎn)生的抗體具有重要的保護(hù)性作用。為此本研究旨在制備出抗弓形蟲(chóng)的人源抗體,為弓形蟲(chóng)病的被動(dòng)免疫防治策略的發(fā)展和應(yīng)用提供理論和實(shí)驗(yàn)依據(jù)。 首先利用基因重組技術(shù)構(gòu)建弓形蟲(chóng)速殖子表面蛋白SAG1的表達(dá)質(zhì)粒,通過(guò)原核表達(dá)系統(tǒng)進(jìn)行大量的表達(dá),并純化制備出具有生物活性的重組SAG1,作為抗體庫(kù)篩選抗原。分離弓形蟲(chóng)病患者的外周血淋巴細(xì)胞,利用工程抗體技術(shù)構(gòu)建出庫(kù)容為3×10的人抗弓形蟲(chóng)免疫球蛋白G基因文庫(kù)。采用克隆印跡法、ELISA、間接免疫熒光反應(yīng)(IFA)等多種方法以重組蛋白對(duì)抗體庫(kù)進(jìn)行較大規(guī)模的篩選驗(yàn)證,對(duì)所得陽(yáng)性克隆進(jìn)行測(cè)序和結(jié)構(gòu)分析。經(jīng)過(guò)6×105個(gè)克隆的篩選,最后得到兩個(gè)陽(yáng)性克隆,分別命名為T(mén)ox08、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(duì)可變區(qū)的劃分,Toxll的輕鏈可變區(qū)部分缺少互補(bǔ)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(duì)Tox08的輕重鏈和Toxll的輕鏈進(jìn)行鏈替換,構(gòu)建鏈替換庫(kù)。經(jīng)篩選后,從Toxll的重鏈與弓形蟲(chóng)病患者來(lái)源的輕鏈庫(kù)構(gòu)建的Toxll輕鏈替換庫(kù)ToxllH-Toxlib中篩選出兩個(gè)陽(yáng)性克隆,分別命名為T(mén)ox87L-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(duì)陽(yáng)性克隆Tox1403L-11H進(jìn)行大量表達(dá),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(duì)其進(jìn)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(píng)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(cè)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(chóng)特異性的人源抗體Fab片段,還同時(shí)確立了其純化方法,為抗弓形蟲(chóng)特異性的人源抗體工廠化生產(chǎn)和臨床研究應(yīng)用提供了理論和實(shí)驗(yàn)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(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
本文編號(hào)：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(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李越希;陶開(kāi)華;張錦海;潘明潔;;弓形蟲(chóng)P30蛋白抗原表位的克隆表達(dá)及純化鑒定[J];中國(guó)生物制品學(xué)雜志;2006年03期
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