抗刚地弓形虫特异性人源抗体Fab片段的制备和研究
发布时间：2019-05-01 06:58
【摘要】：刚地弓形虫(Toxoplasma gondii)简称弓形虫,属于顶复门、真球虫目、弓形虫科,是一种世界性分布的专性细胞内寄生的机会性致病原虫,可寄生于多种脊椎动物的体内。免疫缺损者感染弓形虫后,可导致严重的全身性弓形虫病。可通过母体垂直感染胎儿出现先天性弓形虫病。大量的实验证明在弓形虫感染过程中,机体产生的抗体具有重要的保护性作用。为此本研究旨在制备出抗弓形虫的人源抗体,为弓形虫病的被动免疫防治策略的发展和应用提供理论和实验依据。 首先利用基因重组技术构建弓形虫速殖子表面蛋白SAG1的表达质粒,通过原核表达系统进行大量的表达,并纯化制备出具有生物活性的重组SAG1,作为抗体库筛选抗原。分离弓形虫病患者的外周血淋巴细胞,利用工程抗体技术构建出库容为3×10的人抗弓形虫免疫球蛋白G基因文库。采用克隆印迹法、ELISA、间接免疫荧光反应(IFA)等多种方法以重组蛋白对抗体库进行较大规模的筛选验证,对所得阳性克隆进行测序和结构分析。经过6×105个克隆的筛选,最后得到两个阳性克隆,分别命名为Tox08、Toxll。Tox08和Toxll的重链可变区近似系同为VH3-23,基因同源性分别为98%和98.26%。Tox08的轻链可变区近似系为VK1-33、基因同源性为87.46%；Toxll的轻链可变区近似系为Vκ1-27、基因同源性为93.43%。根据Kabat系统对可变区的划分,Toxll的轻链可变区部分缺少互补决定区1(CDR1),完全缺失CDR2和框架区FR2。为了提高抗体的亲和力,分别对Tox08的轻重链和Toxll的轻链进行链替换,构建链替换库。经筛选后,从Toxll的重链与弓形虫病患者来源的轻链库构建的Toxll轻链替换库ToxllH-Toxlib中筛选出两个阳性克隆,分别命名为Tox87L-11H和Tox1403L-11H。Tox87L-11H和Tox1403L-11H的轻链可变区近似系同为VK：I-17,基因同源性分别为92.11%和89.61%。对阳性克隆Tox1403L-11H进行大量表达,并分别采用抗体亲和层析、钴离子亲和层析和镍离子亲和层析三种纯化方法对其进行纯化。根据纯化后的Fab片段的产量、轻重链比例以及与rSAG1的亲和力评定纯化方法的优劣。经抗体亲和层析和镍离子亲和层析得到的Fab片段的轻重链比例为1：1,产量分别为0.75mg/L和1.08mg/L。钴离子亲和层析纯化的Fab片段产量虽然高于另两种方法,为1.16mg/L,但轻重链的比例为1：2。Biacore检测分析表明,镍离子亲和层析提纯的Fab片段与rSAG1的亲和力最高,其结合常数为9.0×1071/M,解离常数为2.01×10-8M。 上述研究工作不仅制备出具有抗弓形虫特异性的人源抗体Fab片段,还同时确立了其纯化方法,为抗弓形虫特异性的人源抗体工厂化生产和临床研究应用提供了理论和实验依据。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
本文编号：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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 李越希;陶开华;张锦海;潘明洁;;弓形虫P30蛋白抗原表位的克隆表达及纯化鉴定[J];中国生物制品学杂志;2006年03期
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