本文選題：B7-2 + 單鏈抗體��； 參考：《蘇州大學(xué)》2009年碩士論文

【摘要】： 鼠源性單抗在人體使用后可產(chǎn)生人抗鼠抗體(Human Anti-Mouse Antibody, HAMA)反應(yīng),而且分子量大,不能有效進(jìn)入病灶部位,臨床使用受到限制。單鏈抗體(single chain antibody, ScFv)是由一條連接肽將IgG分子重鏈和輕鏈的可變區(qū)連接而成,減少了免疫原性,分子小,實體組織穿透力強,血漿半衰期短,因而在臨床疾病的診斷和治療中具有重要的應(yīng)用價值。 在成功建立穩(wěn)定分泌鼠抗人B7-2雜交瘤細(xì)胞株基礎(chǔ)上,擴增并克隆出該單克隆抗體的重鏈(VH)和輕鏈(VL)可變區(qū)基因。通過重疊延伸PCR(SOE-PCR)方法,在VH和VL可變區(qū)基因之間引入連接肽(Gly4Ser)3,體外構(gòu)建抗人B7-2單鏈抗體(B7-2 ScFv)基因。為便于表達(dá)產(chǎn)物的純化,在抗人B7-2 ScFv的C端增加了6×His tag序列。將其克隆至表達(dá)載體pET32a并在大腸桿菌中進(jìn)行原核表達(dá)。SDS-PAGE和Western-blot分析結(jié)果表明,抗人B7-2 ScFv在BL21(DE3)中獲得高效表達(dá),表達(dá)量占菌體總蛋白30%以上,表達(dá)產(chǎn)物以不溶性包涵體形式存在。經(jīng)過溶解包涵體,體外復(fù)性過程和鎳柱親和層析,純化了抗人B7-2 ScFv。本研究在國內(nèi)首次獲得了高純度的抗人B7-2 ScFv,為今后抗人B7-2ScFv的活性和功能研究以及抗腫瘤藥物的開發(fā)奠定了基礎(chǔ)。
[Abstract]:Human anti-mouse antibody human anti-mouse Antibody (Hama) reaction can be produced after human use of murine monoclonal antibody, and its molecular weight is too large to enter the lesion effectively, so its clinical use is restricted. Single chain antibody (scFv) is a linked peptide that connects the variable regions of the heavy and light chains of IgG molecules, reducing immunogenicity, small molecules, strong penetration of solid tissues, and short plasma half-life. Therefore, it has important application value in the diagnosis and treatment of clinical diseases. On the basis of the successful establishment of stable secreted mouse hybridoma cell lines against human B7-2, the variable region genes of heavy chain (VH) and light chain (VLV) of the monoclonal antibody were amplified and cloned. By means of overlapping extension of PCR- SOE-PCR, the conjugated peptide Gly4Serv3 was introduced between VH and VL variable region gene to construct anti-human B7-2 scFv3 single chain antibody (scFv3) gene in vitro. In order to purify the expressed product, 6 脳 his tag sequence was added to the C-terminal of anti-human B7-2 scFv. The recombinant plasmid pET32a was cloned into E. coli and expressed in E. coli. SDS-PAGE and Western-blot analysis showed that anti-human B7-2 scFv was highly expressed in BL21DDE3, and the expression amount was more than 30% of the total bacterial protein. The expression product existed as insoluble inclusion body. The anti-human B7-2 scFv was purified by dissolution of inclusion body, in vitro renaturation process and affinity chromatography of nickel column. In this study, high purity anti-human B7-2 scFvwas obtained for the first time in China, which laid a foundation for the study of the activity and function of anti-human B7-2ScFv and the development of anti-tumor drugs in the future.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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