本文選題：噬菌體表面展示 + 干擾素α��； 參考：《中國(guó)疾病預(yù)防控制中心》2009年博士論文

【摘要】：系統(tǒng)性紅斑狼瘡(Systemic lupus erythematosus,SLE)是一種嚴(yán)重威脅人類(lèi)健康的自身免疫疾病,研究表明人干擾素α (huIFN-α)與該疾病的發(fā)生有密切關(guān)系,這使其成為SLE臨床治療中重要的靶點(diǎn)分子,通過(guò)設(shè)計(jì)藥物分子阻斷IFN-α和受體結(jié)合,有望起到緩解和治療SLE的目的。 由于huIFN-α是一種自身免疫原,在健康人體內(nèi)不能刺激人產(chǎn)生抗體,因此不能構(gòu)建免疫抗體庫(kù)來(lái)篩選基因工程抗體。本研究以噬菌體表面展示技術(shù)為平臺(tái),從人源全合成抗體庫(kù)中針對(duì)人干擾素α1b (huIFN-α1b)和α2b (huIFN-α2b)篩選抗人干擾素α的基因工程單鏈抗體,具體實(shí)驗(yàn)包括3個(gè)部分： 一、抗人干擾素抗體的篩選、表達(dá)與鑒定 針對(duì)高純度的人干擾素huIFN-α1b蛋白分子,采用高通量的富集篩選策略,從2×109庫(kù)容量的全合成人源抗體庫(kù)中挑選了3000個(gè)克隆,其中20%針對(duì)IFNα1b呈陽(yáng)性反應(yīng),有86株經(jīng)phage ELISA驗(yàn)證對(duì)IFNα-1b, IFNα-2b,γ-干擾素,BSA抗原的反應(yīng),只對(duì)IFN α1b有特異性結(jié)合。通過(guò)對(duì)抗體的CDR3區(qū)的序列分析,根據(jù)抗體在CDR3的長(zhǎng)度和序列上都不同,分別屬于9個(gè)完全不同的單鏈抗體基因,其中有7株抗體輕重鏈可變區(qū)序列分類(lèi)在VH3和VL3家族,有2株抗體輕重鏈可變區(qū)序列分類(lèi)在VH3和VL1家族。通過(guò)phage-ELISA對(duì)9株噬菌體單鏈抗體的結(jié)合特異性和穩(wěn)定性進(jìn)行驗(yàn)證,結(jié)果獲得了5株穩(wěn)定且特異針對(duì)huIFN-α1b而與huIFN-α2b和huIFN-γ無(wú)交叉反應(yīng)的人源單抗。 將5株噬菌粒載體上的抗體基因克隆到scFv單鏈抗體原核表達(dá)載體pET22b,單鏈抗體蛋白通過(guò)pel序列引導(dǎo),分泌到細(xì)菌的周質(zhì)腔中,對(duì)周質(zhì)腔中的scFv抗體蛋白采用金屬螯合層析技術(shù)進(jìn)行分離、純化,從600ml培養(yǎng)基中得到了4mg以上的scFv單鏈抗體。通過(guò)ELISA和Western blot對(duì)5株單鏈抗體的結(jié)合特異性進(jìn)行鑒定,得到3株與huIFN-α1b結(jié)合而與huIFN-α2b和huIFN-γ無(wú)交叉反應(yīng)的人源單抗。 將抗體的輕鏈和重鏈基因克隆到pAc-K-CH3載體系統(tǒng),在桿狀病毒/昆蟲(chóng)細(xì)胞表達(dá)系統(tǒng)中表達(dá)全抗體蛋白,最終通過(guò)免疫熒光和SDS-PAGE分析,證明在桿狀病毒/昆蟲(chóng)細(xì)胞表達(dá)系統(tǒng)中,全抗體基因在昆蟲(chóng)細(xì)胞中高效分泌表達(dá)。表達(dá)的IgG全抗體蛋白采用proteinA親和層析進(jìn)行純化,從1L培養(yǎng)基中得到20mg純度在90%以上的純化的全抗體蛋白。進(jìn)一步通過(guò)ELISA和Western blot對(duì)全抗體的結(jié)合特異性進(jìn)行鑒定,得到3株與huIFN-α1b結(jié)合而與huIFN-α2b和huIFN-γ無(wú)交叉反應(yīng)的人源全抗體蛋白。 二.抗體的表位、親和力和功能活性鑒定 采用競(jìng)爭(zhēng)ELISA對(duì)3株抗體與抗原結(jié)合表位的相互關(guān)系進(jìn)行鑒定,結(jié)果表明,全抗體AIFNal IgG1能夠與3株單鏈抗體AIFNalscFv1、AIFNalscFv2、 AIFNalscFv3競(jìng)爭(zhēng)huIFN-α1b的表位,表明3株抗體與抗原h(huán)uIFN-α1b的結(jié)合表位存在相互重疊的關(guān)系。采用競(jìng)爭(zhēng)ELISA對(duì)3株全抗體與一株具有廣泛結(jié)合人工型干擾素的鼠源單抗4G10與抗原抗原h(huán)uIFN-α1b結(jié)合表位的相互關(guān)系進(jìn)行鑒定,結(jié)果表明,3株全抗體與鼠源單抗4G1d在抗原h(huán)uIFN-α1b的結(jié)合位置上存在重疊的區(qū)域,間接提示3株抗體也具有中和干擾素生物學(xué)功能的活性。采用BIAcore對(duì)抗體與抗原的結(jié)合活性的測(cè)定表明,這幾株抗體(AIFNal IgG1、 AIFNal IgG2和AIFNal IgG3)與huIFN-α1b的親和力KD分別為74.7×10-9、5.44×10-9、11.4×10-9,與干擾素的親和力可以達(dá)到干擾素與受體的親和力水平,為幾株高親和力的抗體。候選了受huIFN-α調(diào)控的基因ISG15和IFIT-1,采用Real-time PCR(實(shí)時(shí)定量PCR)驗(yàn)證干擾素.alb與其抗體在下游信號(hào)通路上的作用,結(jié)果表明在4小時(shí)時(shí)就能檢測(cè)到抗體對(duì)ISG15和IFIT-1的表達(dá)有下調(diào)作用,且這種下調(diào)作用有劑量依賴(lài)效應(yīng),證明了抗體AIFNal IgG1能夠阻斷huIFN-α對(duì)下游基因ISG15和IFIT-1的誘導(dǎo),間接說(shuō)明了抗體是有功能活性的抗體。為了研究抗體所針對(duì)的抗原的功能表位,我們采用WISH-VSV系統(tǒng),按微量細(xì)胞病毒抑制法進(jìn)行了干擾素抗病毒活性中和實(shí)驗(yàn),結(jié)果表明,3株抗體(AIFNal IgG1、AIFNal IgG2和AIFNal IgG3)在濃度達(dá)到200ug/ml時(shí)不能中和干擾素抗病毒活性,而對(duì)照鼠源單抗4G10在濃度達(dá)到100ug/ml時(shí)即能夠中和干擾素抗病毒活性,這從抗體的生物學(xué)活性方面說(shuō)明抗體所針對(duì)huIFN-α1b表位不是干擾素發(fā)揮抗病毒活性的表位。 三.抗體-抗原相互作用的研究 為了研究抗原-抗體的相互作用關(guān)系,我們將干擾素基因克隆pET30a,在大腸桿菌細(xì)胞中表達(dá)了人干擾素蛋白huIFN-α1b,表達(dá)的蛋白通過(guò)Western Blotting鑒定抗原表位。通過(guò)在干擾素基因上引入缺失突變,證明IFNalb的LoopAB區(qū)(29-35)在抗原-抗體相互作用中起有關(guān)鍵的作用,對(duì)該區(qū)域與相鄰區(qū)域的氨基酸的定點(diǎn)突變和westernblot分析表明,IFN的27位的S,33位的D,34位的R,35的H,36位的D突變后,抗體與抗原的結(jié)合可減弱到不可檢測(cè)的水平,為抗原-抗體相互作用的關(guān)鍵的氨基酸殘基。以已有的干擾素a2b結(jié)晶的X-ray衍射3D結(jié)構(gòu)模型為模板(huIFN-α1b和huIFN-α2b的序列相似性為83%),在Discovery Studio(DS)圖像模擬系統(tǒng)中用protein moldeling程序構(gòu)建了干擾素alb分子的模型。以抗體結(jié)構(gòu)模型2A9M(2A9M和AIFNalscFvl的序列相似性為77%)為骨架,在Discovery Studio(DS)圖像模擬系統(tǒng)中搭建完整抗體蛋白的結(jié)構(gòu)模型,對(duì)得到的結(jié)構(gòu)采用Chamm程序?qū)α?chǎng)進(jìn)行優(yōu)化,得到抗體能量最低的結(jié)構(gòu)模型。在Discovery Studio(DS)圖像模擬系統(tǒng)中,采用Z-dock程序,在高速的服務(wù)器上完成抗原-抗體結(jié)構(gòu)的對(duì)接。分析對(duì)接的試驗(yàn)結(jié)果,參照RMSD、 E_Rdock等參數(shù),得到了比較合理的對(duì)接試驗(yàn)結(jié)果,并且這一結(jié)果與表位研究的結(jié)果一致。 綜上所述,本研究運(yùn)用噬菌體表面展示技術(shù),在國(guó)際上首次從人源全合成抗體庫(kù)中成功獲得了3株特異性針對(duì)人干擾素alb的高親和力人源基因工程單克隆抗體,其中有一株通過(guò)體外實(shí)驗(yàn)證明具有免疫調(diào)節(jié)的作用,并首次采用分子建模與表位圖譜鑒定相結(jié)合的方法對(duì)干擾素α、干擾素a受體和干擾素抗體的結(jié)構(gòu)關(guān)系進(jìn)行了研究,對(duì)抗體的功能活性從結(jié)構(gòu)學(xué)上進(jìn)行了解析。本研究為緩解和治療SLE帶來(lái)了希望,對(duì)研究干擾素結(jié)構(gòu)和功能的關(guān)系具有重要意義,為SLE分子機(jī)理的研究提供了基礎(chǔ)。
[Abstract]:Systemic lupus erythematosus ( SLE ) is a kind of autoimmune disease which threatens human health seriously . The study shows that human interferon 偽 ( huIFN - 偽 ) is closely related to the occurrence of the disease , which makes it an important target molecule in SLE clinical treatment .

Human interferon 偽1b ( huIFN - 偽1b ) and 偽2b ( huIFN - 偽2b ) were screened against human interferon 偽1b ( huIFN - 偽1b ) and 偽2b ( huIFN - 偽2b ) .

1 . Screening , expression and identification of anti - human interferon antibodies

A high - throughput screening strategy of human interferon huIFN - 偽1b was used to select 3000 clones from the total synthetic human antibody library of 2 脳 109 cubage .

The antibody gene of 5 strains of phage particles was cloned into the prokaryotic expression vector pET22b of scFv single - chain antibody . Single - chain antibody protein was secreted into the periplasm cavity of bacteria through pel sequence . The scFv antibody protein in periplasm was isolated and purified by metal chelate chromatography . The binding specificity of 5 single - chain antibody was identified by ELISA and Western blot .

The light and heavy chain genes of the antibody were cloned into pAc - K - CH3 vector system . The whole antibody protein was expressed in baculovirus / insect cell expression system .

II . Identification of epitopes , affinity and functional activity of antibodies

In order to study the binding activity of monoclonal antibodies against human interferon - 偽1b , the results showed that the binding sites of the three antibodies ( AIFNal IgG1 , AIFN偽 , IgG3 ) and huIFN - 偽1b could not neutralize the antiviral activity of IFN - 偽1b . The results showed that the antibodies ( AIFNal IgG1 , AIFNal , and AIFNal IgG3 ) could not neutralize the antiviral activity of IFN - 偽1b .

III . Studies on the Interaction of Antibody - antigen

In order to study the interaction between antigen and antibody , we cloned pET30a and expressed human interferon protein huIFN - 偽1b in E . coli cells .

In conclusion , we have successfully obtained three monoclonal antibodies specific to human interferon alb from human - source all - synthetic antibody library by using phage surface display technology . One of them has been proved to be immune - regulated by in vitro experiments , and the structural relationship between interferon - 偽 , interferon - a receptor and interferon antibody is studied by molecular modeling and epitope map identification for the first time . The study provides a basis for studying the relationship between interferon - 偽 , interferon - a receptor and interferon antibody , which is of great significance to study the relationship between interferon structure and function .

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Measurement of scFv antibody affinity using non-competitive enzyme-linked immunosorbent assay[J];Journal of Nanjing Medical University;2005年02期

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本文編號(hào)：1813959