本文選題：噬菌體表面展示 + 人源性單克隆抗體　； 參考：《天津醫(yī)科大學(xué)》2012年博士論文

【摘要】：背景和目的Graves病患者血清中的促甲狀腺素受體抗體(TRAb)分為:(1)TSH受體刺激性抗體(TSAb);(2)TSH受體刺激阻斷性抗體(TSBAb);(3)中性TSH受體抗體。隨著基因工程技術(shù)的發(fā)展,運(yùn)用噬菌體展示技術(shù),通過(guò)對(duì)人源性促甲狀腺素抗體Fab片段抗體庫(kù)的構(gòu)建篩選、鑒定,得到TRAb單克隆抗體,為進(jìn)一步建立TRAb的定量分析方法及探索免疫干預(yù)治療奠定基礎(chǔ)。方法(1)從血清TRAb濃度大于40IU/L的自身免疫性甲狀腺疾病患者外周血中,抽提總RNA,反轉(zhuǎn)錄獲取cDNA。以獲取的cDNA為模版,PCR法擴(kuò)增免疫球蛋白分子輕鏈λ、λ基因及重鏈Fd基因。將輕鏈克隆入pComb3Hss載體以構(gòu)建輕鏈文庫(kù)。將重鏈基因載入κ/λ-pComb3Hss載體,構(gòu)建完成組合文庫(kù)。(2)將重組質(zhì)粒轉(zhuǎn)化大腸桿菌XL1-Blue,用輔助噬菌體M13K07感染,隨機(jī)的組合文庫(kù)表達(dá)于絲狀噬菌體表面,完成噬菌體表面展示。(3)應(yīng)用固相化抗原(TSHR-N)吸附篩選法通過(guò)數(shù)輪"吸附—洗脫—擴(kuò)增"富集噬菌體抗體,篩選陽(yáng)性克隆。(4)取陽(yáng)性克隆的噬菌體DNA,切除gⅢ基因片段,自連接后轉(zhuǎn)化大腸桿菌XL1-B1ue,以IPTG誘導(dǎo)表達(dá)可溶性TRAb Fab片段,應(yīng)用間接ELISA法對(duì)表達(dá)產(chǎn)物進(jìn)行鑒定。(5)利用親和層析柱純化人源性TRAb Fab片段,Western blotting鑒定純化產(chǎn)物。(6)對(duì)表達(dá)較好的陽(yáng)性克隆進(jìn)行測(cè)序分析,輕鏈和重鏈可變區(qū)的DNA序列采用雙向測(cè)序。結(jié)果(1)從外周血單個(gè)核細(xì)胞中抽提得到總RNA,并成功反轉(zhuǎn)錄獲得cDNA文庫(kù)。(2)PCR法擴(kuò)增了大小均為680bp左右的輕鏈κ、λ基因及重鏈Fd基因,并成功構(gòu)建了庫(kù)容為1.32 × 105基因抗體庫(kù)(輕鏈庫(kù))和庫(kù)容為2.28 × 1 05Fab抗體庫(kù)(組合文庫(kù))。(3)通過(guò)噬菌體表面展示技術(shù),在輔助噬菌體M13K07的幫助下,在組合文庫(kù)的基礎(chǔ)上獲得了富含TRAb Fab片段的人源性噬菌體抗體庫(kù)。(4)以TSHR-N為抗原,通過(guò)固相化抗原吸附篩選法對(duì)構(gòu)建的Fab噬菌體抗體庫(kù)進(jìn)行5輪富集篩選,初步成功篩選到特異性TRAb Fab噬菌體抗體,富集效應(yīng)約77倍。(5)通過(guò)Phage-ELISA檢測(cè),結(jié)果鑒定出了具有抗原結(jié)合活性的單克隆。提取陽(yáng)性克隆的噬菌體DNA,切除gⅢ基因片段,實(shí)現(xiàn)了可溶性人源性TRAb Fab片段的表達(dá)。(6)利用Ni2+金屬敖合層析法獲得純化的人源性TRAb Fab片段。(7)間接ELISA法檢測(cè)結(jié)果顯示:制備的可溶性人源性TRAb Fab片段具有特異性結(jié)合TSHR-N端的免疫學(xué)活性。(8)經(jīng)GenBank檢索DNA序列分析,證實(shí)該克隆的輕鏈可變區(qū)與人的免疫球蛋白λ鏈同源性達(dá)到94.4%,重鏈可變區(qū)與人的免疫球蛋白IgG重鏈VH4同源性為88.9%。結(jié)論本研究通過(guò)噬菌體展示技術(shù)成功構(gòu)建了人源性TRAb Fab片段組合文庫(kù),并通過(guò)固相化抗原吸附篩選法篩選獲得陽(yáng)性克隆,實(shí)現(xiàn)了可溶性TRAb Fab片段的表達(dá),基因測(cè)序證實(shí)其輕重鏈與人免疫球蛋白可變區(qū)具有同源性,ELISA結(jié)果顯示其具有特異性結(jié)合TSHR-N端的免疫學(xué)活性。
[Abstract]:Background and objective Thyrotropin receptor (TSH) receptor antibody in the serum of patients with Graves's disease is divided into two groups: TSAB receptor stimulative antibody (TSAB) and TSH receptor stimulating blocking antibody (TSB). With the development of genetic engineering technology, TRAb monoclonal antibody was obtained by using phage display technology and the construction and identification of human thyrotropin antibody Fab fragment antibody library. To establish the quantitative analysis method of TRAb and to explore the immunological intervention therapy. Methods 1) Total RNAs were extracted from peripheral blood of patients with autoimmune thyroid disease whose serum TRAb concentration was higher than 40IU/L, and cDNA was obtained by reverse transcription. The immunoglobulin molecular light chain 位, 位 gene and heavy chain FD gene were amplified by using the obtained cDNA as template. The light chain was cloned into pComb3Hss vector to construct the light chain library. The heavy chain gene was loaded into 魏 / 位 -pComb3Hss vector, and the recombinant plasmid was transformed into Escherichia coli XL1-Blue. the recombinant plasmid was randomly expressed on the surface of filamentous phage by phage M13K07 infection. The phage surface display was completed. (3) the phage DNA of positive clone was screened by solid phase antigen (TSHR-N) adsorption screening method, and the g 鈪，

本文編號(hào)：1977040