全人源抗鼻咽癌噬菌体单链抗体库的构建与初步筛选

发布时间：2018-03-04 23:01

本文选题：EB病毒转化　切入点：噬菌体抗体库　出处：《中南大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：构建全人源抗鼻咽癌噬菌体单链抗体库，从抗体库中筛选抗鼻咽癌的噬菌体单链抗体(ScFv)，并对其进行鉴定和测序分析。方法：体外致敏并用EBV转化鼻咽癌患者的PBMC。用PCR分别扩增VH和VL基因并组成ScFv基因。将ScFv基因与载体fuse5连接后，电击转化大肠杆菌MC1061，构建噬菌体呈现型ScFv库。用人鼻咽癌细胞系CNE2及人胚肺成纤维细胞系HFF对初级噬菌体抗体库进行亲和富集phage-ELISA筛选。筛选获得的阳性克隆进行ELISA及免疫组化鉴定并测序。结果：经EBV转化的3例鼻咽癌患者PBMC，ELISA检测均有抗鼻咽癌抗体产生，经多次PCR，扩增出6种VH(γ、μ)和9种VL(κ、λ)基因，经连接组成54种ScFv基因。将ScFv基因与载体连接后，导入大肠杆菌MC1061。经四环素抗性筛选，得到库容量为6.5×10~7的初级噬菌体抗体库，噬菌体DNA中全长ScFv基因的插入率为100％。用人鼻咽癌细胞系CNE2和人胚肺成纤维细胞系HFF对抗体库进行三轮正负淘选和富集后，从中随机挑取828个克隆进行ELISA筛选，得到245个阳性克隆，阳性率为29.6％。将245个阳性克隆进一步进行其他细胞系的ELISA鉴定，发现克隆F20与三株鼻咽癌细胞系CNE2、HNE2和CNE1均呈阳性反应，而与人胚肺成纤维细胞系HFF、骨髓间质干细胞系HBMSC、原代培养的脐静脉内皮细胞HUVEC及其他人肿瘤细胞系反应呈弱阳性或不反应。免疫细胞化学结果与细胞ELISA结果基本一致；免疫组织化学结果显示：与10例人鼻咽癌组织呈不同程度阳性反应，，反应部位主要见于胞膜、胞浆，而与5例正常鼻黏膜组织均无阳性反应。对克隆F20进行测序分析，结果表明：克隆F20的重链可变区基因与人胚系IgVH3-23有92.8％的同源性，轻链可变区基因与人胚系IgV4-2有94.2％的同源性；V-D-J分别属于VH3-23-D6-6-JH6-linker-V4-2-JL2。结论：应用体外抗原致敏方法和EBV转化技术联合噬菌体抗体库技术，构建库容量达6.5×10~7的全人源抗鼻咽癌噬菌体单链抗体库。通过ELISA、免疫细胞化学和免疫组织化学鉴定，获得的噬菌体抗体克隆F20具有较强的特异性，为鼻咽癌的早期诊断及导向治疗奠定了基础。
[Abstract]:Aim: to construct a full-human phage scFvv library against nasopharyngeal carcinoma and to screen scFvv antibody against nasopharyngeal carcinoma (NPC) from the phage scFvv antibody library, and to identify it and analyze it by sequencing. Methods: PBMC of NPC patients was sensitized and transformed with EBV in vitro. VH and VL genes were amplified by PCR and ScFv genes were constructed. The ScFv gene was ligated with the vector fuse5. The phage display ScFv library was constructed by electroporation of E. coli MC1061. The human nasopharyngeal carcinoma cell line CNE2 and human embryonic lung fibroblast cell line HFF were screened by affinity enrichment phage-ELISA for primary phage antibody library. ELISA and immunohistochemistry were used to identify and sequence. Results: Anti-NPC antibodies were detected by Elisa in 3 NPC patients transformed by EBV. Six VH- (纬, 渭) and nine VL( 魏, 位) genes were amplified by Elisa, and 54 ScFv genes were ligated. The ScFv gene was linked to the vector. After screening for tetracycline resistance, the primary phage antibody library with a capacity of 6.5 脳 10 ~ (7) was obtained. The insertion rate of full-length ScFv gene in phage DNA was 1000.After three rounds of positive or negative panning and enrichment of human nasopharyngeal carcinoma cell line CNE2 and human embryonic lung fibroblast cell line HFF, 828 clones were randomly selected for ELISA screening. 245 positive clones were obtained, and the positive rate was 29.6.The ELISA analysis of other cell lines showed that the clone F20 was positive with three nasopharyngeal carcinoma cell lines CNE2HNE2 and CNE1. The results of immunocytochemistry were consistent with the results of ELISA. The primary cultured umbilical vein endothelial cells (HUVEC) and other human tumor cell lines were weakly positive or unresponsive to human embryonic lung fibroblasts and bone marrow mesenchymal stem cell line HBMSC. the results of immunocytochemistry were similar to those of ELISA cells. The results of immunohistochemistry showed that the positive reaction was in different degree with 10 cases of human nasopharyngeal carcinoma, the reaction site was mainly in the cell membrane and cytoplasm, but no positive reaction was found with 5 cases of normal nasal mucosa. The clone F20 was sequenced. The results showed that the heavy chain variable region gene of the cloned F20 had 92.8% homology with the human embryo line IgVH3-23, and the light chain variable region gene had 94.2% homology with the human embryo line IgV4-2. The V-D-J belonged to VH3-23-D6-JH6-linker-V4-2-JL2, respectively. Conclusion: using in vitro antigen-sensitizing method and EBV transformation technique combined with phage antibody library technique, a full-human anti-nasopharyngeal carcinoma phage scFv library with a capacity of 6.5 脳 10 ~ (7) was constructed. The library was identified by Elisa, immunocytochemistry and immunohistochemistry. The obtained phage antibody clone F20 has strong specificity, which lays a foundation for early diagnosis and guided therapy of nasopharyngeal carcinoma.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R739.63

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