抗乙肝病毒人源噬菌体单链抗体库的构建及筛选

为了构建抗乙肝病毒人源噬菌体特异性单链抗体库,从中筛选抗乙肝病毒人源噬菌体单链抗体库特异性单链抗体,试验提取患者外周淋巴细胞总RNA,以总RNA为模板,通过RT-PCR技术分别扩增出H链和L链可变区基因,采用SOE-PCR方法将VH和VL片段随机拼接成ScFv片段,然后将ScFv片段克隆至pCANTAB5E载体,电转E.coil TG1,构建抗乙肝病毒人源单链抗体库,并从中筛选阳性克隆抗体。结果表明:经过5轮筛选,获得2株能与乙肝病毒抗原特异性结合的阳性克隆。说明利用噬菌体抗体技术可不经免疫制备抗乙肝病毒人源噬菌体特异性单链抗体。

Construction and screening of phage-displayed human single-chain antibody library against Hepatitis B virus

To construct phage-displayed human single-chain variable fragment(ScFv)antibody gene library associated with hepatitis B virus(HBV).Total RNA was extracted from peripheral blood lymphocytes of patients with chronic hepatitis B,and was used to amplify VH and VL gene by RT-PCR.The gene fragments of VH and VL were joined by a DNA linker by SOE-PCR to form the single-chain variable fragments.The ScFvs were coloned into the phage vector pCANTAB5E and the recombinant phagemids were transformed to susceptible Escherichia coli TG1 by electroporation.The results showed that the two strains of positive clones were obtained in the five rounds of screening,which could combine with HBV-specific antigens.It concludes that without immunity,it is feasible to obtain the phage-displayed human single-chain antibody against HBV.