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目的通过噬菌体展示技术构建人源抗恶性疟原虫单链抗体库(ScFv),从中筛选出高亲和力的抗体基因片段,为进一步单链抗体表达打下基础。方法收集患者新鲜外周血,进行淋巴细胞分离和总RNA的提取,提取总RNA经RT-PCR扩增出人源抗体重链和轻链抗体库,采用用重叠延伸拼接法将VH与VL以Linker相连组装成单链抗体基因(ScFv),将ScFv基因片段连接至载体pCANTAB5E,转化大肠杆菌TG 1感受态细胞,构建的鼠源性抗恶性疟原虫天然噬菌体抗体库。结果从20个噬菌体克隆中筛选到15个具有弓形虫可溶性抗原结合活性的阳性克隆。结论从外周血淋巴细胞中获取可变区基因,利用噬菌体抗体库技术制备人源抗恶性疟原虫单链抗体的策略是可行的。
Objective To construct human anti-Plasmodium falciparum single-chain antibody library (ScFv) by phage display technique and select high-affinity antibody gene fragments to lay the foundation for further single-chain antibody expression. Methods Fresh peripheral blood was collected from patients for lymphocyte isolation and total RNA extraction. The total RNA was extracted by RT-PCR to amplify human antibody heavy chain and light chain antibody library. The overlap extension splicing method was used to link VH and VL to Linker Linked to the single chain antibody gene (ScFv), the ScFv gene fragment was ligated to the vector pCANTAB5E and transformed into E. coli TG1 competent cells to construct a natural phage antibody library against murine Plasmodium falciparum. Results Fifteen positive clones with Toxoplasma gondii soluble antigen-binding activity were screened from 20 phage clones. Conclusion The strategy of obtaining variable region genes from peripheral blood lymphocytes and using phage antibody library technology to prepare human anti-Plasmodium falciparum single-chain antibody is feasible.