全人源抗肝癌噬菌体单链抗体库的构建与筛选鉴定

Construction and Screening of Human Anti-hepatoma Single-chain Fv Fusion Phage Libraries

体外致敏并用EBV转化肝癌患者的PBMC.用PCR分别扩增VH和VL基因并组成ScFv基因.将ScFv基因与载体fuse5连接后,电击转化大肠杆菌MC1061,构建噬菌体呈现型ScFv库.用人肝癌细胞及人肝细胞对初级噬菌体抗体库进行亲和富集及phage-ELISA筛选.筛选获得的阳性克隆进行ELISA及免疫组化鉴定并测序.结果表明,经EBV转化的4例肝癌患者PBMC,ELISA检测均有抗肝癌抗体产生,经多次PCR,扩增出6种VH(γ、μ)和9种VL(κ、λ)基因,经连接组成54种ScFv基因.将ScFv基因与载体连接后,导入大肠杆菌MC1061,得到库容为1.0×108的初级噬菌体抗体库,全长ScFv基因的插入率为80%.用人肝癌细胞系HepG2和人肝细胞系QSG-7701对抗体库进行三轮正负淘选和富集后,从中随机挑取533个克隆进行ELISA筛选,得到179个阳性克隆,阳性率为33.6%.将179个阳性克隆进一步进行其他细胞系的鉴定,发现克隆A82对肝癌细胞系HepG2、人胚肾上皮细胞系HEK293呈强阳性反应,免疫组织化学结果显示与人肝细胞癌组织有特异性反应,而不与正常肝组织反应.且A82的相对亲和力为2.4mol/L,解离常数Kd为5.32×10-9mol/L,显示其亲和力较高.对克隆A82进行测序分析,结果表明:A82全长742bp,含linkercDNA序列45bp,重链可变区基因与人胚系IgVH3-23有94.3%的同源性,轻链可变区基因与人胚系IgV4-2有94.9%的同源性,V-D-J分别属于VH3-23-D2-21-JH6-linker-V4-2-JL2.由上述结果可见,应用体外抗原致敏方法和EBV转化技术联合噬菌体抗体库技术,构建了库容量达1×108的全人源抗肝癌单链抗体库,通过细胞ELISA和免疫细胞化学鉴定,获得的噬菌体抗体克隆A82具有较强的特异性,为肝癌的临床诊断及导向治疗奠定了基础.

To construct and screen fully human anti-hepatoma single-chain Fv fusion phage libraries, peripheral blood mononuclear cells （PBMCs） of patients with liver cancer were sensitized in vitro and transformed by Epstein-Barr virus （EBV）. VH and VL genes were reamplified by PCR and combined to single-chain fragment of variable region （ScFv） genes. ScFv genes were cloned into vector fuse5 and transformed into MC1061 by electroporation to construct the ScFv-displaying phage library. The library was subjected to three rounds of positive and negative cell panning and enrichment, then was secreened by phage-ELISA. The binding specificity of phage antibodies with hepatoma carcinoma cells was confirmed by immunohistochemistry with cultured cells and tissue sections. Detection of ELISA showed that 4 liver cancer patients＇B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell. 6 types of VH genes and 9 types of VL genes were obtained by PCR reamplification then connected with （Gly4Ser）3 linker to form 54 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector fuse5 and transformed into MC1061 via electroporation. Phage antibody library with sink size being 1.0×10^8 was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 80%. The library was subjected to three rounds of positive and negative cell panning and enrichment, then was selected by phage-ELISA. After primacy test by HepG2 cell ELISA,179 clones of phage antibody with positive ELISA reaction were picked out of 533 clones.The percentage of positive clones was 33.6%.Though further screened by a panel of cultured cells ELISA,the clone A82 was found to react strongly with HepG2, HEK293, but not with other human tumor cell lines.It also reacted weakly with human hepatic cell line QSG-7701. The results of immunohistochemistry with cultured cells were same as the results of ELISA. A82 was further analyzed after the DNA sequencing.The sequence of A82 was identical. The length of A82 was 742 bp. The VDJ regions of A82 belonged to VH3-23-D2-21-JH6-linker-V4-2-JL2. It can be concluded that fully human anti-hepatoma single-chain Fv fusion phage libraries with sink size being 1.0×10^8 was constructed by means of phage antibody library technique in combination with in vitro immunization method and EBV transformation technique. By cell ELISA and immunohistochemistry with cultured cells and tissue sections, the clone A82 was confirmed to specific bind with hepatoma carcinoma cells.The ScFv fragment against hepatoma may be further developed and applied to clinical diagnosis and therapy.