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目的 :构建噬菌体人源抗独特型抗体库。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基因与载体连接后 ,转化大肠杆菌MC10 6 1,构建噬菌体呈现型ScFv库。结果 :经EBV转化的 10例鼻咽癌患者的PBMC中 ,8例有鼻咽癌抗独特型抗体产生。经多次PCR ,扩增出 5种VH(γ、μ)和 7种VL(κ、λ)基因 ,经连接组成 14种ScFv基因。将ScFv基因与载体连接后 ,导入大肠杆菌MC10 6 1。经四环素抗性筛选 ,得到库容为 1.1× 10 7的初级噬菌体抗独特型抗体库 ,噬菌体DNA中全长ScFv基因的插入率为 70 %。结论 :用体外致敏法结合噬菌体抗体库技术 ,制备人源抗独特型单链抗体(Ab2 βScFv)的策略是可行的
Objective: To construct phage human anti-idiotypic antibody library. Methods: PBMCs from patients with nasopharyngeal carcinoma were sensitized in vitro and transformed with EBV. The VH and VL genes were amplified by PCR and the ScFv genes were constructed. The ScFv gene was ligated with the vector and transformed into E. coli MC10 6 1 to construct a phage-displayed ScFv library. RESULTS: Of the 10 PBMCs from EBV-transformed NPC patients, 8 had nasopharyngeal carcinoma anti-idiotypic antibodies. After multiple PCR, five kinds of VH (γ, μ) and seven kinds of VL (κ, λ) genes were amplified and linked to form 14 kinds of ScFv genes. The ScFv gene was ligated with the vector and introduced into E. coli MC10 6 1. After tetracycline resistance screening, a primary phage anti-idiotypic antibody library with a library volume of 1.1 × 10 7 was obtained. The insertion rate of the full-length ScFv gene in the phage DNA was 70%. Conclusion: The strategy of preparing human anti-idiotypic single chain antibody (Ab2βScFv) by in vitro sensitization combined with phage antibody library technology is feasible