人源抗恶性疟原虫单链抗体库的制备及鉴定

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。

Preparation and identification of a human single chain Fv antibody against Plasmodium falciparum

OBJECTIVE To construct humanize phage antibody library against Plasmodium falciparum and to screen specificity scFv to Plasmodium falciparum.METHODS Total RNA was extracted from fresh blood of patients with Plasmodium falciparum followed RT-PCR.The heavy-chain and light-chain variable region genes(VH and VL)repertoire of immunoglobulin were amplified respectively by PCR.Then they were linked as VH-linker-VL sequence forming scFv by SOE(splicing by overlap extension)PCR.These fragments were cloned into the phagemid pCANTAB5E and the recombinant phagemids were transformed to susceptible Escherichi a coli TGl by roporation.After rescuing with helper phage M13K07,A natural single-chain antibody library for Plasmodium falciparum was successfully constructed.RESULTS 15 antigen binding clones were identified from 20 individual phagemid clones.CONCLUSION This study shows the strategy of phage display technique and of RT-PCR from peripheral blood lymphocytes mRNA may provide an alternative approach.