本文選題：人源化 + 單鏈抗體�。� 參考：《暨南大學(xué)》2007年博士論文

【摘要】：目的：在已篩選獲得1株高親和力特異性抗肝癌單鏈抗體(scFv) DM的基礎(chǔ)上,采用結(jié)合蛋白質(zhì)同源模建技術(shù)、互補(bǔ)決定區(qū)(CDR)移植技術(shù)和噬菌體展示技術(shù)的抗體庫優(yōu)化法,同步實(shí)現(xiàn)人源化和優(yōu)化非人源氨基酸殘基,以獲得特異性強(qiáng)、親和力高、免疫原性低的人源化抗肝癌scFv,為肝癌的早期診治提供新的方法與手段。 方法：通過計(jì)算機(jī)輔助,采用同源模建技術(shù)模建抗肝癌scFv DM的三維結(jié)構(gòu)；通過BLAST,從GenBank中選擇與scFv DM的重鏈(VH)和輕鏈(VL)同源性最高的人源抗體序列為人源化模板；通過分子結(jié)構(gòu)分析和序列分析,確定對(duì)抗原結(jié)合位點(diǎn)的構(gòu)象有重要影響的骨架區(qū)(FR)殘基,把難以判斷回復(fù)突變殘基的人、鼠源副本同時(shí)編入人源化抗體序列中；通過重疊延伸PCR技術(shù)合成人源化抗體全基因,利用噬菌體展示技術(shù)構(gòu)建人源化組合抗體庫；通過肝癌細(xì)胞篩選陽性克隆,ELISA方法測(cè)定各個(gè)克隆抗原結(jié)合活性；用硫氰酸鹽洗脫法測(cè)定并比較親本抗體和人源化scFv的相對(duì)親和力。 結(jié)果：抗肝癌scFvDM的三維結(jié)構(gòu)示：六個(gè)CDR襻位于Fv段的N端,形成一個(gè)與抗原結(jié)合的口袋,口袋深16.1A。用Prostat驗(yàn)證,模建的三維結(jié)構(gòu)的79.8%的Φ和Ψ分布于Ramachandran圖的核心區(qū)域,模板1NQB的為83.2%；確定人抗體序列AAW67378和BAB18257分別作為VH和VL人源化模板,FR同源性分別為76.1%和71.6%；確定人、鼠VH和VL的FR序列分別有20個(gè)和23個(gè)不同殘基,其中28個(gè)人源化殘基、8個(gè)回復(fù)突變殘基和7個(gè)難以確定殘基；成功構(gòu)建人源化組合抗體庫,抗體庫包含了由25不同重鏈和22不同輕鏈組成的128種人源化DM；經(jīng)過篩淘及ELISA鑒定,獲得HDM1、HDM2和HDM3三個(gè)陽性克隆,相對(duì)親和力指數(shù)分別為：HDM1 1.6mol/L、HDM2 1.2mol/L。HDM3 2.2mol/L 結(jié)論：成功模建抗肝癌scFv DM的三維結(jié)構(gòu),模擬結(jié)構(gòu)合理可靠；抗體庫優(yōu)化法是一個(gè)高效、簡(jiǎn)便的人源化方法；獲得的3株陽性克隆HDM1、HDM2、HDM3與親本抗體DM同樣具有良好的抗原結(jié)合特異性和較高的親和力,為進(jìn)一步探索在肝癌早期診療的臨床應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Objective: on the basis of screening a high affinity specific single chain antibody (scFvDM) against hepatocellular carcinoma (HCC), an antibody library optimization method was used to optimize the antibody library by using the technique of protein homology modeling, complementary determinant region (CDR) transplantation and phage display.The humanization and optimization of non-human amino acid residues were carried out simultaneously to obtain the humanized anti-HCC scFv with strong specificity, high affinity and low immunogenicity, which provided a new method and means for the early diagnosis and treatment of HCC.Methods: the three-dimensional structure of anti-HCC scFv DM was constructed by computer aided modeling, and the human antibody sequence with the highest homology to scFv DM and light chain GenBank was selected as the humanized template by BLAST.Through molecular structure analysis and sequence analysis, the skeleton FR) residues which have important influence on the conformation of antigen-binding sites were determined.The whole humanized antibody gene was synthesized by overlapping extension PCR technique, the human combined antibody library was constructed by phage display technique, and the binding activity of each clone antigen was determined by Elisa method for screening positive clones of hepatoma cells.The relative affinity of parental antibody and humanized scFv was determined and compared by thiocyanate elution method.Results: the three-dimensional structure of anti-hepatoma scFvDM showed that six CDR loops were located at the N-terminal of the FV segment, forming an antigen-binding pocket with a depth of 16.1 A.By Prostat, 79.8% of 桅 and 蠄 were distributed in the core region of Ramachandran map and 83.2% of template 1NQB. The homology of human antibody sequence AAW67378 and BAB18257 as VH and VL humanized template FR were 76.1% and 71.6%, respectively.The FR sequences of mouse VH and VL have 20 and 23 different residues, of which 28 are humanized residues, 8 are reverse-mutated residues and 7 are difficult to determine the residues.The antibody library contains 128 kinds of humanized DMs composed of 25 different heavy chains and 22 different light chains. After screening and ELISA identification, three positive clones of HDM1, HDM2 and HDM3 were obtained, and the relative affinity index was 1.6 mol / L HDM2 1.2mol/L.HDM3 2.2mol/L, respectively.Conclusion: the three-dimensional structure of anti-hepatoma scFv DM was successfully constructed, and the simulated structure was reasonable and reliable, and the antibody library optimization method was an efficient and simple humanization method.The three positive clones, HDM1, HDM2HDM3, also had good antigen-binding specificity and high affinity with their parental antibody DM, which laid a foundation for further exploration of clinical application in the early diagnosis and treatment of liver cancer.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊冬華,畢向軍,農(nóng)玉新;抗肝癌單鏈抗體基因庫的制備[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2001年12期

2 ;Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen[J];Hepatobiliary & Pancreatic Diseases International;2006年02期

3 武國(guó)軍,郝曉柯;單鏈抗體的研究進(jìn)展及在腫瘤診斷、治療中的應(yīng)用[J];國(guó)外醫(yī)學(xué)(臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè));1999年01期

4 袁清安,俞煒源,孔德清,賀秉坤;血管內(nèi)皮生長(zhǎng)因子特異性鼠源單鏈抗體的人源化[J];生物技術(shù)通訊;2001年02期

5 汪保安,陳曉穗,王欲曉,曲佳,周麗君,王琰;通過隨機(jī)突變提高抗TNF-α單鏈抗體的親和力[J];細(xì)胞與分子免疫學(xué)雜志;2005年04期

6 黃u&,壽成超;噬菌體抗體庫技術(shù)的研究進(jìn)展[J];細(xì)胞生物學(xué)雜志;2002年01期

7 王字玲,胡遠(yuǎn)東,焦克芳,李松,榮康泰;梭曼單鏈抗體酶EP_6一級(jí)結(jié)構(gòu)的確定和三維結(jié)構(gòu)的模建[J];中國(guó)藥理學(xué)與毒理學(xué)雜志;2000年05期

8 周e，

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