本文選題：噬菌體抗體庫 + 單鏈抗體　； 參考：《天津大學》2012年博士論文

【摘要】：抗體是體液免疫系統(tǒng)的重要組成部分，也在生命科學研究、疫病檢測和醫(yī)藥領域發(fā)揮著重要作用。噬菌體展示技術是人源治療性抗體的重要技術來源之一。TNF-α是一種前炎癥因子，與風濕性關節(jié)炎等多種疾病的發(fā)生密切相關。本研究采用噬菌體抗體展示技術構建一個人源噬菌體抗體庫，并從中篩選出具有TNF-α細胞毒活性抑制作用的人源單鏈抗體。 依據(jù)文獻報道的人抗體恒定區(qū)序列及人源抗體各胚系基因序列，本研究設計42條引物，以200份人外周血中分離的總RNA為模板，利用這些引物擴增出了抗體的重鏈（VH）和輕鏈（VL）可變區(qū)基因。抗體重鏈可變區(qū)的擴增和組裝采用三輪OE-PCR反應完成：首先分別擴增VH的CDR1、CDR2、CDR3和FR3，然后采用OE-PCR拼接重鏈的CDR1和CDR2、CDR3和FR3的基因片段，再將兩者拼接成完整的人抗體VH基因。VL基因的擴增采用PCR方法從總RNA中經(jīng)多次反應獲得，再將其與VH基因連接形成完整的scFv基因。 噬菌體庫的構建采用載體pCANTAB5E、E.coli TG1和輔助性噬菌體M13KO7。將scFv基因插入pCANTAB5E載體酶切位點Sfi I和Not I之間，并將新載體轉化E.coli TG1。輔助噬菌體M13KO7侵染轉化后的TG1，，構建scFv噬菌體庫。梯度稀釋法測得噬菌體庫庫容為2.5×10~7，噬菌體滴度為10~(12)pfu�；驕y序顯示E.coli TG1中轉化DNA與預期相符，抗體重鏈各區(qū)域組合正確，VH和VL之間有柔性多肽編碼基因相連。采用微孔板篩選法以人TNF-α、禽流感NS1蛋白、人血清白蛋白HSA、牛血清白蛋白OVA和PRRSV病毒GP5蛋白對文庫進行淘選，結果顯示五種蛋白在淘選過程中均能有效富集。 為獲得高親和力的抗TNF-α抗體，依據(jù)phage ELISA實驗結果從隨機挑選的87株重組噬菌體中篩選出4株陽性克隆進行重組蛋白表達和抗TNF-α活性測試。重組蛋白的表達采用pET-28a載體和大腸桿菌BL21（DE3）菌株。IPTG誘導后SDS-PAGE電泳顯示scFv重組蛋白在大腸桿菌中大量表達，重組蛋白的表達量占菌體量的40%左右，經(jīng)鎳柱純化的蛋白純度在90%以上。MTT法檢測TNF-α殺傷L292細胞的活性，顯示在放線菌素為10μg/mL時，TNF-α的ED50為0.01ng/mL。重組蛋白B11和A24對TNF-α的IC_(50)分別為70μg/mL和100μg/mL。 本研究構建、鑒定了人源scFv噬菌體抗體庫，并從中篩選了2株具有抗TNF-α活性的人源scFv，為抗TNF-α抗體藥物的研發(fā)奠定了基礎。
[Abstract]:Antibody is an important part of humoral immune system, and also plays an important role in life science research, disease detection and medicine. Phage display is one of the important technical sources of human therapeutic antibodies. TNF- 偽 is a proinflammatory factor, which is closely related to the occurrence of many diseases such as rheumatoid arthritis. In this study, a human phage antibody library was constructed by phage antibody display technique, from which human scFv with TNF- 偽 cytotoxic activity was screened. According to the reported sequence of human antibody constant region and human antibody genes, 42 primers were designed, using 200 samples of total RNA isolated from human peripheral blood as template. These primers were used to amplify the VH (heavy chain) and VLV (light chain) variable region genes of antibodies. Three rounds of OE-PCR reaction were used to amplify and assemble the variable region of heavy chain of antibody. First, CDR1, CDR2, CDR3 and FR3 of VH were amplified, and then the CDR1 and CDR2 CDR3 and FR3 fragments of heavy chain were spliced by OE-PCR. The VH gene. VL gene of human antibody was amplified from total RNA by PCR method. The VH gene was ligated with VH gene to form a complete scFv gene. The phage library was constructed by vector pCANTAB5 E.coli TG1 and auxiliary bacteriophage M13KO7. The scFv gene was inserted into the pCANTAB5E vector between Sfi I and Not I, and the new vector was transformed into E.coli TG1. The scFv phage library was constructed by assisting the phage M13KO7 to infect the transformed TG1. The phage library capacity was 2.5 脳 10 ~ (7) and the phage titer was 10 ~ (10) ~ (10) ~ (12) pfuu by gradient dilution method. Gene sequencing showed that the transformed DNA in E.coli TG1 was in accordance with expectations, and the correct combination of regions of antibody heavy chain showed that there was a flexible polypeptide coding gene link between VH and VL. Human TNF- 偽, avian influenza NS1 protein, human serum albumin (HSA), bovine serum albumin (BSA) OVA and PRRSV virus GP5 protein were screened by microplate screening method. The results showed that the five proteins were effectively enriched during panning. In order to obtain anti-TNF- 偽 antibody with high affinity, four positive clones were screened from 87 randomly selected recombinant phages according to the results of phage ELISA assay to detect the expression of recombinant protein and the activity of anti-TNF- 偽. The expression of recombinant protein was induced by pET-28a vector and E. coli BL21DE3 strain. SDS-PAGE electrophoresis showed that the recombinant scFv protein was expressed in a large amount in E. coli, and the expression of recombinant protein was about 40% of the total number of bacteria. The activity of TNF- 偽 in killing L292 cells was detected by MTT assay. The results showed that the ED50 of TNF- 偽 was 0.01 ng / mL when actinomycin was 10 渭 g/mL. The ICs of recombinant protein B11 and A24 for TNF- 偽 were 70 渭 g/mL and 100 渭 g / mL, respectively. In this study, the human scFv phage antibody library was constructed, and two human scFvs with anti-TNF- 偽 activity were screened, which laid a foundation for the development of anti-TNF- 偽 antibody drugs.
【學位授予單位】：天津大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

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