本文選題：EB病毒轉(zhuǎn)化　切入點：噬菌體抗體庫　出處：《中南大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的：構建全人源抗鼻咽癌噬菌體單鏈抗體庫，從抗體庫中篩選抗鼻咽癌的噬菌體單鏈抗體(ScFv)，并對其進行鑒定和測序分析。 方法：體外致敏并用EBV轉(zhuǎn)化鼻咽癌患者的PBMC。用PCR分別擴增VH和VL基因并組成ScFv基因。將ScFv基因與載體fuse5連接后，電擊轉(zhuǎn)化大腸桿菌MC1061，構建噬菌體呈現(xiàn)型ScFv庫。用人鼻咽癌細胞系CNE2及人胚肺成纖維細胞系HFF對初級噬菌體抗體庫進行親和富集phage-ELISA篩選。篩選獲得的陽性克隆進行ELISA及免疫組化鑒定并測序。 結果：經(jīng)EBV轉(zhuǎn)化的3例鼻咽癌患者PBMC，ELISA檢測均有抗鼻咽癌抗體產(chǎn)生，經(jīng)多次PCR，擴增出6種VH(γ、μ)和9種VL(κ、λ)基因，經(jīng)連接組成54種ScFv基因。將ScFv基因與載體連接后，導入大腸桿菌MC1061。經(jīng)四環(huán)素抗性篩選，得到庫容量為6.5×10~7的初級噬菌體抗體庫，噬菌體DNA中全長ScFv基因的插入率為100％。用人鼻咽癌細胞系CNE2和人胚肺成纖維細胞系HFF對抗體庫進行三輪正負淘選和富集后，從中隨機挑取828個克隆進行ELISA篩選，得到245個陽性克隆，陽性率為29.6％。將245個陽性克隆進一步進行其他細胞系的ELISA鑒定，發(fā)現(xiàn)克隆F20與三株鼻咽癌細胞系CNE2、HNE2和CNE1均呈陽性反應，而與人胚肺成纖維細胞系HFF、骨髓間質(zhì)干細胞系HBMSC、原代培養(yǎng)的臍靜脈內(nèi)皮細胞HUVEC及其他人腫瘤細胞系反應呈弱陽性或不反應。免疫細胞化學結果與細胞ELISA結果基本一致；免疫組織化學結果顯示：與10例人鼻咽癌組織呈不同程度陽性反應，，反應部位主要見于胞膜、胞漿，而與5例正常鼻黏膜組織均無陽性反應。對克隆F20進行測序分析，結果表明：克隆F20的重鏈可變區(qū)基因與人胚系IgVH3-23有92.8％的同源性，輕鏈可變區(qū)基因與人胚系IgV4-2有94.2％的同源性；V-D-J分別屬于VH3-23-D6-6-JH6-linker-V4-2-JL2。 結論：應用體外抗原致敏方法和EBV轉(zhuǎn)化技術聯(lián)合噬菌體抗體庫技術，構建庫容量達6.5×10~7的全人源抗鼻咽癌噬菌體單鏈抗體庫。通過ELISA、免疫細胞化學和免疫組織化學鑒定，獲得的噬菌體抗體克隆F20具有較強的特異性，為鼻咽癌的早期診斷及導向治療奠定了基礎。
[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- (緯, 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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392;R739.63

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