大容量人源肝癌核糖体展示单链抗体库的构建

发布时间：2018-03-09 12:28

  本文选题：肝癌　切入点：核糖体展示技术　出处：《兰州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的： 构建人源肝癌天然库容量大、多样性好的核糖体展示单链抗体库,为开发治疗性人源抗体奠定良好的实验基础。 方法： 分别收集40例人新鲜外周血,各2ml(肝癌患者10名、健康成人26名、2岁儿童2名、新生儿2名),进行淋巴细胞分离和总RNA的提取,通过设计合适的优化引物用RT-PCR技术扩增人抗体重链可变区基因(variable region of heavy chain,VH)、轻链可变区基因(variable region of light chain,VL)和作为间隔区的轻链恒定区基因(consist region of light chain, CK)。采用改进的重叠延伸PCR (SOEPCR)技术将VH和VL进行拼接,连接肽为Linker(Gly4Ser)3。之后引入构建核糖体展示单链抗体(scFv)库模板所需元件,包括一个T7启动子、一个核糖体结合位点(Kozak序列)和翻译起始密码,以及作为间隔区的CK基因。模板基因片段连接T-Vector转化E.coli DH5a大肠杆菌,对所构建的单链抗体库进行菌落PCR鉴定和测序分析。 结果： 1、构建了库容量为2.65×1013的人源肝癌单链抗体库。 2、经菌落PCR鉴定和测序分析阳性重组子的序列,我们发现该抗体库具有丰富的多样性。 结论： 采用改进的重叠延伸PCR (SOE-PCR)方法,对构建库容量高、多样性好的人源性肝癌核糖体展示单链抗体库,提高了建库效率。成功构建的抗体库,为后续筛选抗体、抗体修饰等工作打下扎实的实验基础。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392;R735.7
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本文编号：1588552