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目的:利用细胞内重组的方法,构建大库容量的人源性抗SARS病毒单链抗体(scFv)库,为筛选SARS病毒抗原的人源性抗体奠定基础。方法:取6例SARS康复患者的80mL外周血,分离淋巴细胞后提取总RNA。分离纯化mRNA并反转录成cDNA后,扩增抗体重链、轻链可变区基因片段。然后经重叠延伸PCR装配成scFv基因并克隆入噬粒载体pDAN5中,电转化大肠杆菌TG1构建初级抗体库。制备初级库噬菌体后,感染大肠杆菌BS1365进行细胞内重组制备次级抗体库。结果:初级库库容量为3×109,在大肠杆菌BS1365中重组后得到3×1011的次级抗体库。结论:细胞内重组方法的应用可使大库容量抗体库的构建变得简单易行。构建的抗SARS病毒单链抗体次库不仅接近人类天然抗体库的水平,而且多样性好,为筛选抗SARS病毒抗原的抗体提供了保证。
OBJECTIVE: To construct a large library of human anti-SARS single chain antibody scFv by means of intracellular recombination, and to lay a foundation for the screening of human antibodies against SARS virus antigen. Methods: Totally 80 mL peripheral blood from 6 SARS patients were collected. Total RNA was extracted after lymphocytes were isolated. Isolation and purification of mRNA and reverse transcription into cDNA, amplification of antibody heavy chain, light chain variable region gene fragments. Then, the scFv gene was assembled by overlap extension PCR and cloned into the phagemid vector pDAN5. The primary antibody library was constructed by electrotransformation into E. coli TG1. After the primary library phage was prepared, E. coli BS1365 was infected for intracellular recombination to prepare a secondary antibody library. Results: The primary pool volume was 3 × 109, and 3 × 1011 secondary antibody pools were obtained after recombination in E. coli BS1365. Conclusion: The application of intracellular recombination method makes the construction of large library of antibody capacity easy. The construction of the single-chain antibody against SARS virus sub-pool not only close to the level of human natural antibody library, and diversity, and screening for anti-SARS virus antigens provided an assurance.