本文選題：熱帶傳染病 + 快速診斷　； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：熱帶傳染病是威脅我軍指戰(zhàn)員身體健康的重要疾病。部隊(duì)官兵熱帶傳染病自我防護(hù)意識(shí)比較薄弱且進(jìn)入新環(huán)境因缺乏免疫力等而極易感染熱帶傳染病。由于部隊(duì)執(zhí)行多樣化的軍事任務(wù),迫切需要建立熱帶傳染病偵檢、診治和防控相關(guān)技術(shù),應(yīng)對(duì)熱帶傳染病的威脅。瘧疾屬于蟲媒傳染病,由瘧原蟲寄生人體紅細(xì)胞引起,被WHO列入的8大熱帶病之一,廣泛分布于熱帶亞熱帶地區(qū),且近年來(lái)瘧原蟲對(duì)抗瘧藥普遍具有抗藥性,潛在危害極大。恙蟲病是由恙蟲病東方體感染人體而引起的蟲媒傳染病,近年來(lái)在我國(guó)的流行區(qū)域有不斷擴(kuò)大延伸的趨勢(shì)。我國(guó)南海島嶼地處熱帶,常年多雨,溫暖潮濕,是各種病原體滋生的溫床,有調(diào)查顯示該地區(qū)大部分區(qū)域是恙蟲病的疫源地。由于駐守海島的部隊(duì)多為外來(lái)人群,對(duì)該類傳染病的免疫力低,發(fā)病率較高,嚴(yán)重影響了部隊(duì)各項(xiàng)任務(wù)的完成,是我國(guó)南方戰(zhàn)區(qū)造成非戰(zhàn)斗減員的重要傳染病。部隊(duì)軍事作業(yè)及戰(zhàn)時(shí)的特點(diǎn)都要求建立快速、便捷、無(wú)需或較少借助儀器的診斷方法。為了建立這樣一種滿足現(xiàn)場(chǎng)使用要求的熱帶傳染病快速偵檢方法,我們引入了基因工程雙特異性抗體BsAb,它是將兩種針對(duì)不同抗原的單克隆抗體A和B的重、輕鏈可變區(qū)以非共價(jià)鍵、共價(jià)鍵或亮氨酸拉鏈、螺旋-轉(zhuǎn)角-螺旋等蛋白質(zhì)結(jié)構(gòu)域連接形成的重組抗體分子。利用基因工程雙特異性抗體可同時(shí)結(jié)合兩種不同的抗原的特性,如構(gòu)建抗人紅細(xì)胞/抗病原體抗原的雙特異性抗體,該BsAb可同時(shí)分別結(jié)合人紅細(xì)胞和血液中病原體,形成可見(jiàn)的紅細(xì)胞的集聚,從而對(duì)病原體感染做出診斷檢測(cè)。本課題在制備了抗惡性瘧原蟲裂殖子表面抗原、抗恙蟲病東方體外膜抗原以及抗人紅細(xì)胞膜表面非凝集抗原的單克隆抗體雜交瘤細(xì)胞的基礎(chǔ)上,通過(guò)RT-PCR分別獲得cDNA,以其為模板,利用設(shè)計(jì)的多對(duì)引物PCR擴(kuò)增得到各單克隆抗體的重鏈和輕鏈可變區(qū)目的基因片段VH、VL。設(shè)計(jì)拼接引物,利用重疊延伸PCR法,將抗瘧原蟲裂殖子表面抗原的單克隆抗體的重鏈和輕鏈可變區(qū)目的基因片段通過(guò)連接肽序列拼接得到抗-瘧原蟲裂殖子表面抗原的單鏈抗體scFv目的基因片段;將抗人紅細(xì)胞膜表面非凝集抗原的單克隆抗體的重鏈和輕鏈可變區(qū)目的基因片段與抗恙蟲病東方體外膜抗原的單克隆抗體的重鏈和輕鏈可變區(qū)目的基因片段通過(guò)兩個(gè)短連接肽序L10(SAKTTPKLGG)和一個(gè)長(zhǎng)連接肽序列LL(RADAAAA(G4S)4)拼接得到抗-人紅細(xì)胞膜表面非凝集抗原/抗-恙蟲病東方體外膜抗原的雙特異性抗體BsAb目的基因片段。同時(shí)分別在其N端引入畢赤酵母真核表達(dá)系統(tǒng)中信號(hào)肽前導(dǎo)序列,在其C端引入6×His標(biāo)簽序列。將這些基因片段分別克隆至畢赤酵母分泌表達(dá)載體pPIC9K,電轉(zhuǎn)化畢赤酵母菌GS115并誘導(dǎo)表達(dá),先通過(guò)SDS-PAGE電泳分析確定疑似目的蛋白條帶后,再應(yīng)用Western blot檢測(cè)目的蛋白的表達(dá)。獲得的確定有目的蛋白表達(dá)的上清后續(xù)可經(jīng)鎳柱純化得到電泳純重組抗體。本研究中,我們通過(guò)重疊延伸PCR獲得抗-瘧原蟲裂殖子表面抗原的單鏈抗體scFv和抗-人紅細(xì)胞膜表面非凝集抗原/抗-恙蟲病東方體外膜抗原的雙特異性抗體BsAb目的基因片段。將抗-瘧原蟲裂殖子表面抗原的單鏈抗體scFv克隆至畢赤酵母分泌表達(dá)載體pPIC9K上進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE和Western blot檢測(cè)分析重組蛋白的表達(dá),得到的重組蛋白大小與理論分子量相符。間接免疫熒光實(shí)驗(yàn)結(jié)果表明該單鏈抗體可與瘧原蟲裂殖子表面蛋白識(shí)別并結(jié)合,從而檢測(cè)到瘧原蟲感染,具有與親本抗體相似的識(shí)別結(jié)合能力。將抗-人紅細(xì)胞膜表面非凝集抗原/抗-恙蟲病東方體外膜抗原的雙特異性抗體重組基因片段克隆至畢赤酵母真核分泌表達(dá)載體pPIC9K進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE和Western blot檢測(cè)重組蛋白的表達(dá),得到的重組蛋白大小與理論分子量相符。紅細(xì)胞集聚實(shí)驗(yàn)結(jié)果表明該雙特異性抗體具有與紅細(xì)胞膜表面非凝集抗原識(shí)別并結(jié)合的活性,為后續(xù)將該基因工程雙特異性抗體用于恙蟲病等感染性疾病病原體的快速診斷以及種屬鑒定等提供技術(shù)支撐。
[Abstract]:Tropical infectious diseases are the main disease threatening the health of the army. The troops of self - protection awareness is relatively weak and tropical infectious diseases into the new environment because of the lack of immunity and vulnerable to tropical infectious diseases. Because the troops carry out diversified military tasks, the urgent need to establish a tropical infectious disease detection, diagnosis and prevention and control technology to deal with the threat of infectious diseases. Tropical malaria belongs to insect borne diseases caused by Plasmodium parasites in human red blood cells, one of the 8 major tropical diseases were included in the WHO, is widely distributed in tropical and subtropical regions, and in recent years, Plasmodium antimalarial drugs generally have drug resistance, the potential harm is great. Tsutsugamushi disease is infectious disease by tsutsugamushi Oriental body disease caused by the infection of human, in recent years in epidemic areas in our country has the expanding trend in the South China Sea. The island is located in the tropics, perennial rainy, warm and moist, is A hotbed of various pathogens, a survey shows that most of this area is the focus of tsutsugamushi disease. As for foreign troops stationed in the island population, the infectious disease is low, high incidence rate, serious impact on the military tasks, is an infectious disease in South China caused by the non combat attrition theater an important characteristic of the military operation. And wartime are required to establish a fast, convenient, without or less using instrument diagnostic method. In order to establish such a satisfying tropical infectious disease rapid detection method using the site requirements, I have introduced the engineered bispecific antibody BsAb, it is two different antigens monoclonal antibody A and B heavy and light chain variable region by non covalent bonds, covalent bond or leucine zipper, recombinant antibody molecule protein domain helix turn helix connection. The use of gene Bispecific antibody can be combined with the engineering characteristics of two different antigens, such as the construction of bispecific antibody against human red blood cell / pathogen antigen, the BsAb can also bind to the pathogen of human red blood cells respectively and blood, the formation of agglomeration visible red blood cells, so as to make a diagnosis of pathogen infection detection in this article. The preparation of Plasmodium falciparum merozoite surface antigen, anti Orientia outer membrane antigen and anti human RBC membrane surface agglutination antigen monoclonal antibody hybridoma cells, cDNA were obtained by RT-PCR with the template, using several pairs of primers designed by PCR was amplified by heavy chain monoclonal antibody and the light chain variable region gene fragment VH, VL. splicing design primers, using overlap extension PCR method, the heavy chain of monoclonal antibodies against Plasmodium falciparum merozoite surface antigen and a light chain variable region objective base Because the fragment by connecting peptide sequence scFv scFv gene fragment of anti Plasmodium merozoite surface antigen; the purpose of the heavy chain and light chain variable region gene fragment to the heavy chain of monoclonal antibody against human erythrocyte membrane surface agglutination antigen and a light chain variable region gene fragment with anti Orientia membrane resistance the original monoclonal antibody by two short peptide sequence L10 (SAKTTPKLGG) and a peptide sequence of LL long connection (RADAAAA (G4S) 4) spliced anti human erythrocyte membrane surface agglutination antigen / anti Orientia membrane antigen bispecific antibody BsAb fragment respectively. At the same time the N is introduced into Pichia pastoris eukaryotic expression system of signal peptide in its preamble sequence, C is introduced into 6 * His tag sequence. These gene fragments were cloned into Pichia pastoris expression vector pPIC9K secretion, electroporation of Pichia yeast Strain GS115 and induced by SDS-PAGE electrophoresis analysis, to determine the suspected target protein bands, the expression and application of the target protein was detected by Western blot. The determination of the target protein expression was subsequent purification by Ni column electrophoresis pure recombinant antibody. In this study, we obtained by overlap extension PCR scFv scFv anti merozoite surface antigen and anti human RBC membrane surface agglutination antigen / anti Orientia membrane antigen bispecific antibody BsAb fragment. The anti Plasmodium merozoite surface antigen single chain antibody scFv was cloned into pPIC9K vector for expression of Pichia pastoris, and SDS-PAGE Western blot expression analysis of recombinant protein, the recombinant protein has a molecular mass and size theory. The experimental results show that the indirect immunofluorescence antibody and malaria Insect merozoite surface protein recognize and bind to the detection of Plasmodium infection, thus, with the recognition of similar and parental antibody binding ability. The anti human erythrocyte membrane surface agglutination antigen / anti Orientia membrane antigen bispecific antibody gene fragment was cloned into Pichia pastoris eukaryotic expression vector pPIC9K was induced the expression, expression of recombinant protein SDS-PAGE and Western blot, the recombinant protein with the theoretical molecular weight of red blood cells. The experimental results show that the concentration of bispecific antibody with surface and erythrocyte agglutination antigen recognition and non binding activity, for the follow-up of the gene engineering bispecific antibody is used to provide technical support for rapid diagnosis of tsutsugamushi infectious disease pathogens and species identification.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R824

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 郭文娟;;膠體金檢測(cè)卡與血涂片法在瘧原蟲檢測(cè)中的價(jià)值比較[J];現(xiàn)代醫(yī)藥衛(wèi)生;2014年16期

2 艾國(guó)平;;熱帶醫(yī)學(xué)的起源與發(fā)展趨勢(shì)[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2012年08期

3 龔震宇;楊小平;;全球被忽視熱帶病的防制概況[J];疾病監(jiān)測(cè);2011年05期

4 戚中田;;二十一世紀(jì)熱帶軍事醫(yī)學(xué)[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2010年06期

5 周志成;郝文波;吳英松;章平;郝芬;李明;;瘧原蟲免疫膠體金試劑盒的研制及性能評(píng)價(jià)[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2010年05期

6 汪茂榮;;恙蟲病的流行病學(xué)與診治進(jìn)展[J];東南國(guó)防醫(yī)藥;2009年06期

7 劉敏;王露楠;;噬菌體展示技術(shù)在感染性疾病診斷上的應(yīng)用[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2009年03期

8 方美玉;田小東;;中國(guó)熱帶地區(qū)主要蟲媒病毒病的病原學(xué)及快速檢測(cè)[J];中華流行病學(xué)雜志;2007年12期

9 何偉;朱蔭昌;梁幼生;戴建榮;徐明;唐建霞;曹國(guó)群;華萬(wàn)全;李遠(yuǎn)林;楊忠;;糞檢與免疫診斷方法檢測(cè)日本血吸蟲感染效果比較[J];中國(guó)血吸蟲病防治雜志;2007年02期

10 李卉,潘紀(jì)春,劉子,劉景漢,章金剛;抗人紅細(xì)胞膜抗原非凝集型單克隆抗體的研制及特性鑒定[J];細(xì)胞與分子免疫學(xué)雜志;2005年04期

，

本文編號(hào)：1736853