本文選題：肝癌　切入點(diǎn)：核糖體展示技術(shù)　出處：《蘭州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的： 構(gòu)建人源肝癌天然庫(kù)容量大、多樣性好的核糖體展示單鏈抗體庫(kù),為開(kāi)發(fā)治療性人源抗體奠定良好的實(shí)驗(yàn)基礎(chǔ)。 方法： 分別收集40例人新鮮外周血,各2ml(肝癌患者10名、健康成人26名、2歲兒童2名、新生兒2名),進(jìn)行淋巴細(xì)胞分離和總RNA的提取,通過(guò)設(shè)計(jì)合適的優(yōu)化引物用RT-PCR技術(shù)擴(kuò)增人抗體重鏈可變區(qū)基因(variable region of heavy chain,VH)、輕鏈可變區(qū)基因(variable region of light chain,VL)和作為間隔區(qū)的輕鏈恒定區(qū)基因(consist region of light chain, CK)。采用改進(jìn)的重疊延伸PCR (SOEPCR)技術(shù)將VH和VL進(jìn)行拼接,連接肽為L(zhǎng)inker(Gly4Ser)3。之后引入構(gòu)建核糖體展示單鏈抗體(scFv)庫(kù)模板所需元件,包括一個(gè)T7啟動(dòng)子、一個(gè)核糖體結(jié)合位點(diǎn)(Kozak序列)和翻譯起始密碼,以及作為間隔區(qū)的CK基因。模板基因片段連接T-Vector轉(zhuǎn)化E.coli DH5a大腸桿菌,對(duì)所構(gòu)建的單鏈抗體庫(kù)進(jìn)行菌落PCR鑒定和測(cè)序分析。 結(jié)果： 1、構(gòu)建了庫(kù)容量為2.65×1013的人源肝癌單鏈抗體庫(kù)。 2、經(jīng)菌落PCR鑒定和測(cè)序分析陽(yáng)性重組子的序列,我們發(fā)現(xiàn)該抗體庫(kù)具有豐富的多樣性。 結(jié)論： 采用改進(jìn)的重疊延伸PCR (SOE-PCR)方法,對(duì)構(gòu)建庫(kù)容量高、多樣性好的人源性肝癌核糖體展示單鏈抗體庫(kù),提高了建庫(kù)效率。成功構(gòu)建的抗體庫(kù),為后續(xù)篩選抗體、抗體修飾等工作打下扎實(shí)的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective:. In order to establish a large capacity and good diversity ribosomal display single chain antibody library of human hepatocellular carcinoma (HCC), a good experimental foundation for the development of therapeutic human antibody was established. Methods:. Fresh peripheral blood samples were collected from 40 patients with liver cancer (10 patients with liver cancer, 26 healthy adults, 2 children aged 2 years old and 2 newborns). Lymphocyte isolation and total RNA extraction were performed. The variable region of heavy chain variable region of heavy chain VHG, the variable region of light chain VHV gene and the light chain constant region gene consist region of light chain, CKP were amplified by designing suitable primers. The modified heavy weight was used to amplify the variable region of heavy chain chain VHV gene and the light chain constant-region gene as the spacer region. VH and VL are spliced by PCR / SOEPCR technology. The ligand peptide is Linkerg4Seran 3.After the introduction of the ribosome display scFvvlibrary template, it includes a T7 promoter, a ribosomal binding site, Kozak sequence, and translation initiation code. The CK gene and template gene fragment were linked to T-vector to transform E. coli DH5a Escherichia coli. The constructed scFv library was identified by colony PCR and sequenced. Results:. 1. A single chain antibody library of human hepatocellular carcinoma with a capacity of 2.65 脳 10 13 was constructed. 2. By colony PCR identification and sequencing analysis, we found that the antibody library had rich diversity. Conclusion:. By using the improved overlapping extension PCR SOE-PCR method, the single-chain antibody library with high capacity and high diversity of human hepatocellular carcinoma ribosomes was constructed, and the efficiency of the library was improved. The successfully constructed antibody library was used for the subsequent screening of antibodies. Antibody modification laid a solid experimental foundation.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392;R735.7
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本文編號(hào)：1588552