抗人SARS病毒单链抗体库的构建

Construction of a large human scFv library against SARS virus

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。

AIM: To construct a large human scFv library against SARS virus by using in vivo recombination. METHODS: Total RNA was isolated from the lymphocytes of 6 patients recovered from SARS. mRNA was isolated and reverse transcribed into cDNA. The V_H and V_L fragments were amplified from the cDNA and then assembled into scFv genes. The scFv genes were amplified and ligated into phagemid pDAN5. The primary library was constructed by transforming the recombinant phagemid into E.coli TG1. The secondary library was generated by in vivo recombination in E.coli BS1365 following the infection of BS1365 by primary library phages. RESULTS: A primary library of 3×10 9 and a second library of 3×10 11 were constructed. CONCLUSION: A large human scFv library against SARS virus with good diversity was constructed, which may be used for screening antibodies to SARS virus antigens.