人—兔嵌合型抗弓形虫IgM抗体基因库的构建和抗弓形虫IgG抗体参考物质的研究
发布时间：2019-05-18 01:19
【摘要】：刚地弓形虫(Toxoplasma gondii)是一种可寄生于有核细胞的原虫,能感染人和动物,引发弓形虫病。成年人多为隐形感染,但对于某些特定人群,感染可引起严重的临床症状,这些人群包括孕妇、艾滋病、器官移植、恶性肿瘤患者等。临床实验室弓形虫检测主要依靠免疫血清学检测,检测项目通常包括特异IgM和IgG等。在检测中,为了排除假阴性结果,控制检验操作过程,保证检测结果准确可靠,需要阳性质控品进行室内质量控制和室间质量评价。我们前期的研究采用基因工程抗体技术将鼠的抗弓形虫P30抗原IgG抗体可变区基因与人IgM抗体恒定区基因进行了拼接,在哺乳动物细胞中表达得到了鼠-人嵌合抗弓形虫IgM抗体,对嵌合抗体作为IgM检测质控替代品进行了探索,但是此嵌合抗体仅针对弓形虫P30抗原,不能完全模拟人血清样本中的能针对弓形虫多个抗原多个表位的特异性抗体。针对此问题,本课题中我们拟用弓形虫天然抗原免疫家兔,测定效价后取兔外周血单个核细胞,经淋巴细胞分离液处理后提取总RNA,逆转录获得cDNA,作为模板,分别扩增兔抗弓形虫抗体轻链和重链可变区基因库,分别与人轻链恒定区和重链恒定区基因重组,制备了人-兔嵌合型抗弓形虫IgM抗体轻链和重链基因库,未来其在哺乳动物细胞中表达后可得到多克隆抗体,或可从中进一步筛选出高亲合力的人-兔嵌合基因工程抗体,二者均可作为质控品的替代品。该嵌合型轻、重链基因库的成功克隆和人源化表达质粒的构建为下一步人源化人-兔嵌合型抗弓形虫抗体IgM抗体的表达和高亲合力抗体的筛选打下了基础,并向人-兔嵌合型抗弓形虫抗体IgM作为弓形虫抗体IgM检测质控品的应用迈进了一步。弓形虫特异IgG抗体的免疫血清学检测方法有多种,以酶联免疫法和化学发光方法在临床实验室筛查检测中最为常用。目前我国能提供该检测试剂的厂家有多个,原理基本是间接法。无论采用何种检测方法和试剂,保证在不同实验室、不同‘试剂和方法间检测的结果具有可比性十分重要,这就是临床检验标准化的目的,实现临床检验标准化或结果可比性的重要前提是要有统一的量值溯源。为此,英国国家生物学标准物质和质控物研究所(National Institute for Biological Standards and Control, NIBSC)于2004年研制并提供了人抗弓形虫特异IgG抗体的国际标准品,编号为01/600,含量值为20IU,被世界卫生组织(World Health Organization, WHO)认定为第一代抗弓形虫IgG抗体国际标准物质。然而,国际标准物质不但较难获取,且价格非常昂贵,不适用于我国临床实验室大规模使用,而此类标准物质的研制又是有效开展医疗卫生工作的需要。因此,本研究参照了有关标准,研制了抗弓形虫IgG血清学参考物质,并取得国家一级标准物质证书(编号：GBW09192),建立和应用可溯源到国际标准的我国自己的标准物质。应用该参考物质,将不仅使我国人类抗弓形虫IgG血清学检测结果具有可比性,而且有了溯源性。其应用范围包括：(1)不同试剂、不同检测方法检测下限的确定；(2)依据该参考物质制备的标准系列血清可用作厂家试剂的工作标准；(3)用于临床实验室日常检测的质量控制等。此外,以其为标准制备的质控样本也被我们应用于抗弓形虫IgG抗体检测的室间质量评价,通过室间质量评价结果,临床实验室可了解本实验室结果与其他实验室结果之间的可比性,分析检测中可能存在的实际问题,并采取持续改进措施,又进一步促进了临床检验标准化。
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a kind of protozoa that can be parasitic on nucleated cells, can infect humans and animals, and cause toxoplasmosis. The majority of adults are invisible, but for certain specific populations, infection can cause serious clinical symptoms, including pregnant women, AIDS, organ transplantation, and malignant tumors. The detection of Toxoplasma gondii in the clinical laboratory mainly depends on the detection of the immune serum, and the detection items usually include specific IgM and IgG. In the test, in order to exclude false negative results, control the operation of the test, ensure that the test result is accurate and reliable, and the positive quality control is required for indoor quality control and room-to-room quality evaluation. The mouse-human chimeric anti-toxoplasma IgM antibody was expressed in the mammalian cells by splicing the variable region gene of the anti-Toxoplasma P30 antigen IgG antibody of the mouse and the human IgM antibody constant region gene by using the genetic engineering antibody technology, The chimeric antibody is used as an IgM detection quality control alternative, but the chimeric antibody is only for the P30 antigen of the Toxoplasma gondii, and can not completely simulate the specific antibody which can be used for the multiple epitopes of the toxoplasma gondii in the human serum sample. In order to solve this problem, we use the natural antigen of Toxoplasma gondii to immunize the rabbit, measure the titer, take the peripheral blood mononuclear cells of the rabbit, extract the total RNA after the lymphocyte separation liquid treatment, reverse transcription to obtain the cDNA, as the template, the light chain and the heavy chain variable region gene bank of the rabbit anti-toxoplasma antibody light chain and the heavy chain variable region are respectively amplified, and the human-rabbit chimeric anti-toxoplasma IgM antibody light chain and the heavy chain gene bank are prepared by the recombination of the human light chain constant region and the heavy chain constant region gene, In the future, a polyclonal antibody can be obtained after expression in a mammalian cell, or a human-rabbit chimeric gene engineering antibody with high affinity can be further screened from the human-rabbit chimeric gene engineering antibody, both of which can be used as a substitute for quality control products. The successful cloning of the chimeric light and heavy chain gene library and the construction of the humanized expression plasmid provide a basis for the expression of the next humanized human-rabbit chimeric anti-toxoplasma antibody IgM antibody and the screening of the high-affinity antibody, And a step is taken to the application of the human-rabbit chimeric anti-toxoplasma antibody IgM as a toxoplasma antibody IgM detection quality control product. There are many methods to detect the immune serum of the specific IgG antibody of Toxoplasma gondii, and the enzyme-linked immunosorbent assay and the chemiluminescence method are the most commonly used in the screening and detection of clinical laboratory. At present, there are a number of manufacturers that can provide the detection reagent, and the principle is basically indirect method. Regardless of the detection methods and reagents used, it is important to ensure that the results of the detection between different laboratories, different reagents and methods are of comparable importance, which is the purpose of the standardization of clinical testing, To this end, the National Institute for Biological Standards and Control (NIBSC), the National Institute for Biological Standards and Control (NIBSC), developed and provided international standards for human anti-toxoplasma-specific IgG antibodies in 2004. The number is 01/600, the content is 20 IU, and is recognized by the World Health Organization as the first generation of the international standard substance of the anti-toxoplasma IgG antibody. However, the international standard substance is not only difficult to obtain, and the price is very expensive, and is not applicable to the large-scale use of the clinical laboratory in China, The development of such a standard substance is also a need for effective medical and health work. Therefore, the study has made reference to the relevant standards, developed the anti-Toxoplasma IgG serological reference substance, and obtained the national standard substance certificate (No.: GBW09192). To establish and apply our own standard material which can be traced to international standards. The application of this reference material will not only make the serological test results of the human anti-toxoplasmosis IgG in our country comparable, but also have the traceability. The application range of the reference material includes: (1) different reagents, And (2) the standard series of serum prepared according to the reference substance can be used as the working standard of the manufacturer's reagent; (3) the quality control for the daily detection of the clinical laboratory and the like. The quality control samples prepared as a standard are also used in the room-to-room quality evaluation of the anti-Toxoplasma IgG antibody detection, and the clinical laboratory can understand the comparability between the results of the laboratory and other laboratory results through the results of the room-to-room quality evaluation. The practical problems that may exist in the analysis and detection are analyzed, and the measures of continuous improvement are taken, and the standardization of clinical inspection is further promoted.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
本文编号：2479552
[Abstract]:Toxoplasma gondii (Toxoplasma gondii) is a kind of protozoa that can be parasitic on nucleated cells, can infect humans and animals, and cause toxoplasmosis. The majority of adults are invisible, but for certain specific populations, infection can cause serious clinical symptoms, including pregnant women, AIDS, organ transplantation, and malignant tumors. The detection of Toxoplasma gondii in the clinical laboratory mainly depends on the detection of the immune serum, and the detection items usually include specific IgM and IgG. In the test, in order to exclude false negative results, control the operation of the test, ensure that the test result is accurate and reliable, and the positive quality control is required for indoor quality control and room-to-room quality evaluation. The mouse-human chimeric anti-toxoplasma IgM antibody was expressed in the mammalian cells by splicing the variable region gene of the anti-Toxoplasma P30 antigen IgG antibody of the mouse and the human IgM antibody constant region gene by using the genetic engineering antibody technology, The chimeric antibody is used as an IgM detection quality control alternative, but the chimeric antibody is only for the P30 antigen of the Toxoplasma gondii, and can not completely simulate the specific antibody which can be used for the multiple epitopes of the toxoplasma gondii in the human serum sample. In order to solve this problem, we use the natural antigen of Toxoplasma gondii to immunize the rabbit, measure the titer, take the peripheral blood mononuclear cells of the rabbit, extract the total RNA after the lymphocyte separation liquid treatment, reverse transcription to obtain the cDNA, as the template, the light chain and the heavy chain variable region gene bank of the rabbit anti-toxoplasma antibody light chain and the heavy chain variable region are respectively amplified, and the human-rabbit chimeric anti-toxoplasma IgM antibody light chain and the heavy chain gene bank are prepared by the recombination of the human light chain constant region and the heavy chain constant region gene, In the future, a polyclonal antibody can be obtained after expression in a mammalian cell, or a human-rabbit chimeric gene engineering antibody with high affinity can be further screened from the human-rabbit chimeric gene engineering antibody, both of which can be used as a substitute for quality control products. The successful cloning of the chimeric light and heavy chain gene library and the construction of the humanized expression plasmid provide a basis for the expression of the next humanized human-rabbit chimeric anti-toxoplasma antibody IgM antibody and the screening of the high-affinity antibody, And a step is taken to the application of the human-rabbit chimeric anti-toxoplasma antibody IgM as a toxoplasma antibody IgM detection quality control product. There are many methods to detect the immune serum of the specific IgG antibody of Toxoplasma gondii, and the enzyme-linked immunosorbent assay and the chemiluminescence method are the most commonly used in the screening and detection of clinical laboratory. At present, there are a number of manufacturers that can provide the detection reagent, and the principle is basically indirect method. Regardless of the detection methods and reagents used, it is important to ensure that the results of the detection between different laboratories, different reagents and methods are of comparable importance, which is the purpose of the standardization of clinical testing, To this end, the National Institute for Biological Standards and Control (NIBSC), the National Institute for Biological Standards and Control (NIBSC), developed and provided international standards for human anti-toxoplasma-specific IgG antibodies in 2004. The number is 01/600, the content is 20 IU, and is recognized by the World Health Organization as the first generation of the international standard substance of the anti-toxoplasma IgG antibody. However, the international standard substance is not only difficult to obtain, and the price is very expensive, and is not applicable to the large-scale use of the clinical laboratory in China, The development of such a standard substance is also a need for effective medical and health work. Therefore, the study has made reference to the relevant standards, developed the anti-Toxoplasma IgG serological reference substance, and obtained the national standard substance certificate (No.: GBW09192). To establish and apply our own standard material which can be traced to international standards. The application of this reference material will not only make the serological test results of the human anti-toxoplasmosis IgG in our country comparable, but also have the traceability. The application range of the reference material includes: (1) different reagents, And (2) the standard series of serum prepared according to the reference substance can be used as the working standard of the manufacturer's reagent; (3) the quality control for the daily detection of the clinical laboratory and the like. The quality control samples prepared as a standard are also used in the room-to-room quality evaluation of the anti-Toxoplasma IgG antibody detection, and the clinical laboratory can understand the comparability between the results of the laboratory and other laboratory results through the results of the room-to-room quality evaluation. The practical problems that may exist in the analysis and detection are analyzed, and the measures of continuous improvement are taken, and the standardization of clinical inspection is further promoted.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392
【参考文献】
相关博士学位论文 前1条
1 潘阳;人—鼠嵌合型抗弓形虫IgM抗体检测质控物研制和基于病毒样颗粒的microRNA-146a转运方法建立及其在系统性红斑狼疮中的治疗研究[D];北京协和医学院;2013年
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