本文關(guān)鍵詞： 蘇丹紅I 單鏈Fab抗體 包涵體 酶聯(lián)免疫吸附測定　出處：《華東師范大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 抗體是機(jī)體免疫系統(tǒng)針對侵入體內(nèi)的抗原物質(zhì)所產(chǎn)生的一種特異性免疫球蛋白,抗體研究的發(fā)展經(jīng)歷了多克隆抗體、單克隆抗體和基因工程抗體三個(gè)階段�；蚬こ炭贵w是將抗體基因或基因片段通過基因工程技術(shù)進(jìn)行重組,并在不同表達(dá)系統(tǒng)中產(chǎn)生的抗體或抗體片段,目前基因工程抗體包括人-鼠嵌合抗體、改型抗體、雙特異性抗體、小分子抗體等。 所謂小分子抗體是指相對分子質(zhì)量較小但具有抗原結(jié)合功能的抗體分子片段,包括Fab、Fv、scFv等,其中Fab和scFv是目前小分子抗體研究的重點(diǎn)。 小分子抗體具有制備方法簡單、免疫原性弱、穿透力強(qiáng)等優(yōu)點(diǎn),因此在腫瘤導(dǎo)向治療和病毒性疾病的治療,以及放射免疫成像檢查腫瘤等方面得到了廣泛的應(yīng)用。 本課題是在結(jié)合小分子抗體Fab和scFv優(yōu)缺點(diǎn)的基礎(chǔ)上,擬克隆抗蘇丹紅Ⅰ單克隆抗體的編碼基因Lc和Fd,并將二者在原核表達(dá)載體pET24a內(nèi)用人工合成的Linker連接,構(gòu)建成基因工程抗體scFab的表達(dá)載體,最后通過原核表達(dá),期望能得到一種兼具scFv和Fab優(yōu)點(diǎn)的新型基因工程抗體scFab。本論文主要包括以下幾個(gè)部分: 1.從分泌抗蘇丹紅Ⅰ單克隆抗體的雜交瘤細(xì)胞中通過PCR技術(shù)獲得Lc和Fd基因,克隆至T-A克隆載體后,進(jìn)行克隆鑒定和DNA測序。DNA序列BLAST和編碼蛋白質(zhì)序列分析表明所得到的抗體基因符合小鼠的IgG基因特征。 2.經(jīng)過對已有載體的改造,重新設(shè)計(jì)了可以直接用于scFab原核表達(dá)的載體;同時(shí),在上述對抗體分析的基礎(chǔ)上,選取合適Lc和Fd基因片段,在其兩端引入酶切位點(diǎn)后與人工合成的Linker相連,一起克隆到經(jīng)過改造的載體pET24a(+)中,構(gòu)建成表達(dá)載體pET24a-scFab,成功轉(zhuǎn)染大腸桿菌E.coliBL21(DE3)。 3.IPTG誘導(dǎo)scFab在E.coli BL21(DE3)表達(dá),SDS-PAGE分析和Western Blot鑒定,顯示scFab蛋白以包涵體的形式表達(dá),經(jīng)過對其表達(dá)條件進(jìn)行優(yōu)化后,獲得大量表達(dá)的scFab抗體蛋白包涵體。 4.尿素溶解scFab包涵體,以含谷胱甘肽氧化還原系統(tǒng)的復(fù)性液對其進(jìn)行復(fù)性,透析濃縮得到復(fù)性抗體,間接ELISA和競爭ELISA檢測結(jié)果表明復(fù)性抗體對蘇丹紅Ⅰ具有很高的結(jié)合活性和結(jié)合特異性。 論文結(jié)論:本研究成功構(gòu)建了一種新型基因工程抗體scFab,為新型基因工程抗體研究奠定了初步的理論和實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Antibody is a kind of specific immunoglobulin produced by the body's immune system against the antigen substances invading the body. The development of antibody research has experienced the development of polyclonal antibody. Monoclonal antibody and genetic engineering antibody are three stages. Genetic engineering antibody is the antibody or fragment produced in different expression systems by recombination of antibody gene or gene fragment through genetic engineering technology. At present, genetic engineering antibodies include human-mouse chimeric antibody, modified antibody, bispecific antibody, small molecule antibody and so on. The so-called small molecular antibody refers to the relatively small molecular weight of antibody fragments with antigen-binding function, including FV FV scFv, among which Fab and scFv are the focus of the study of small molecular antibodies. Because of the advantages of simple preparation, weak immunogenicity and strong penetration, small molecular antibodies have been widely used in tumor-oriented therapy, treatment of viral diseases, and radioimmunoimaging. On the basis of combining the advantages and disadvantages of small molecule antibody Fab and scFv, the encoding genes L c and FD of monoclonal antibody against Sudan I were cloned and ligated with synthetic Linker in prokaryotic expression vector pET24a. The expression vector of genetic engineering antibody (scFab) was constructed. Finally, by prokaryotic expression, we hope to obtain a novel genetic engineering antibody scFab. which has the advantages of both scFv and Fab. This thesis mainly includes the following parts:. 1. LC and FD genes were obtained by PCR from hybridoma cells secreting monoclonal antibody against Sudan 鈪，

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