人—兔嵌合型抗弓形蟲IgM抗體基因庫的構建和抗弓形蟲IgG抗體參考物質(zhì)的研究
發(fā)布時間：2019-05-18 01:19
【摘要】：剛地弓形蟲(Toxoplasma gondii)是一種可寄生于有核細胞的原蟲,能感染人和動物,引發(fā)弓形蟲病。成年人多為隱形感染,但對于某些特定人群,感染可引起嚴重的臨床癥狀,這些人群包括孕婦、艾滋病、器官移植、惡性腫瘤患者等。臨床實驗室弓形蟲檢測主要依靠免疫血清學檢測,檢測項目通常包括特異IgM和IgG等。在檢測中,為了排除假陰性結果,控制檢驗操作過程,保證檢測結果準確可靠,需要陽性質(zhì)控品進行室內(nèi)質(zhì)量控制和室間質(zhì)量評價。我們前期的研究采用基因工程抗體技術將鼠的抗弓形蟲P30抗原IgG抗體可變區(qū)基因與人IgM抗體恒定區(qū)基因進行了拼接,在哺乳動物細胞中表達得到了鼠-人嵌合抗弓形蟲IgM抗體,對嵌合抗體作為IgM檢測質(zhì)控替代品進行了探索,但是此嵌合抗體僅針對弓形蟲P30抗原,不能完全模擬人血清樣本中的能針對弓形蟲多個抗原多個表位的特異性抗體。針對此問題,本課題中我們擬用弓形蟲天然抗原免疫家兔,測定效價后取兔外周血單個核細胞,經(jīng)淋巴細胞分離液處理后提取總RNA,逆轉錄獲得cDNA,作為模板,分別擴增兔抗弓形蟲抗體輕鏈和重鏈可變區(qū)基因庫,分別與人輕鏈恒定區(qū)和重鏈恒定區(qū)基因重組,制備了人-兔嵌合型抗弓形蟲IgM抗體輕鏈和重鏈基因庫,未來其在哺乳動物細胞中表達后可得到多克隆抗體,或可從中進一步篩選出高親合力的人-兔嵌合基因工程抗體,二者均可作為質(zhì)控品的替代品。該嵌合型輕、重鏈基因庫的成功克隆和人源化表達質(zhì)粒的構建為下一步人源化人-兔嵌合型抗弓形蟲抗體IgM抗體的表達和高親合力抗體的篩選打下了基礎,并向人-兔嵌合型抗弓形蟲抗體IgM作為弓形蟲抗體IgM檢測質(zhì)控品的應用邁進了一步。弓形蟲特異IgG抗體的免疫血清學檢測方法有多種,以酶聯(lián)免疫法和化學發(fā)光方法在臨床實驗室篩查檢測中最為常用。目前我國能提供該檢測試劑的廠家有多個,原理基本是間接法。無論采用何種檢測方法和試劑,保證在不同實驗室、不同‘試劑和方法間檢測的結果具有可比性十分重要,這就是臨床檢驗標準化的目的,實現(xiàn)臨床檢驗標準化或結果可比性的重要前提是要有統(tǒng)一的量值溯源。為此,英國國家生物學標準物質(zhì)和質(zhì)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蟲特異IgG抗體的國際標準品,編號為01/600,含量值為20IU,被世界衛(wèi)生組織(World Health Organization, WHO)認定為第一代抗弓形蟲IgG抗體國際標準物質(zhì)。然而,國際標準物質(zhì)不但較難獲取,且價格非常昂貴,不適用于我國臨床實驗室大規(guī)模使用,而此類標準物質(zhì)的研制又是有效開展醫(yī)療衛(wèi)生工作的需要。因此,本研究參照了有關標準,研制了抗弓形蟲IgG血清學參考物質(zhì),并取得國家一級標準物質(zhì)證書(編號：GBW09192),建立和應用可溯源到國際標準的我國自己的標準物質(zhì)。應用該參考物質(zhì),將不僅使我國人類抗弓形蟲IgG血清學檢測結果具有可比性,而且有了溯源性。其應用范圍包括：(1)不同試劑、不同檢測方法檢測下限的確定；(2)依據(jù)該參考物質(zhì)制備的標準系列血清可用作廠家試劑的工作標準；(3)用于臨床實驗室日常檢測的質(zhì)量控制等。此外,以其為標準制備的質(zhì)控樣本也被我們應用于抗弓形蟲IgG抗體檢測的室間質(zhì)量評價,通過室間質(zhì)量評價結果,臨床實驗室可了解本實驗室結果與其他實驗室結果之間的可比性,分析檢測中可能存在的實際問題,并采取持續(xù)改進措施,又進一步促進了臨床檢驗標準化。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
本文編號：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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R392
【參考文獻】
相關博士學位論文 前1條
1 潘陽;人—鼠嵌合型抗弓形蟲IgM抗體檢測質(zhì)控物研制和基于病毒樣顆粒的microRNA-146a轉運方法建立及其在系統(tǒng)性紅斑狼瘡中的治療研究[D];北京協(xié)和醫(yī)學院;2013年
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