丙型肝炎病毒非结构蛋白4A人源单链可变区抗体的筛选与鉴定

Screening and characterization of human phage antibody to hepatitis C NS4A antigen

目的利用噬菌体表面展示技 术,从半合成人源化单链可变区抗体库中筛选、鉴定丙型肝炎病毒(HCV)非结构蛋白NS4A 的单 链可变区抗体(ScFv)及其编码基因,为抗HCV的细胞内免疫基因治疗研究开辟新途径。方法采用噬菌体表面展示技术,以重组的HCV非结构蛋白NS4A为包被抗原,从噬菌体单链可变区抗体库中经过3轮“吸附-洗脱-扩增”筛选过 程,获得抗原结合活性较强的HCV NS4A人单链可变区抗体片段阳性克隆,并对其 进行免疫学活性及编码基因序列测定。结果筛选得到 的ScFv片段具有抗HCV NS4A的特异性,编码基因符合人源化单链可变区抗体编码基因的特 点。结论利用噬菌体抗体库技术,成功获得HCV NS4A的特异性人抗体,为进一步研究HCV NS4A的生物学功能及细胞内免疫抗HCV基因治疗方 案奠定基础。

Objective To screen and identify humanized single chain variable region antibody for hepatitis C virus non structural 3 protein. Methods Phage display technique was employed in the screening and identification of humanized single chain variable region(ScFv) antibody by using recombinant hepatitis C virus (HCV) non structural 3 protein as coating antigen. Results After 5 rounds of biopanning of a ScFv antibody phage library, 66 phage clones were evaluated by enzyme linked immunosorbent assay(ELISA).2 phage clones were selected from 66 clones according to the highest OD value in the ELISA identification and lowest cross reaction to the bovine serum albumin(BSA).The coding fragments of 2 ScFv were sequenced. After 5 rounds of biopanning of the ScFv phage library with initial 2.0×10 13 clones, the HCV NS3 antigen binding phage particles have been concentrated by 300 times. 66 phage clones were selected randomly for the subsequent ELISA screening and identification by using HCV NS3 and BSA as coating antigens, respectively. 2 phage clones were selected for their high A value in the ELISA identification and low cross reaction to BSA. The 2 inserts in the phage vector were sequenced and confirmed its ScFv characterization. Conclusion We have successfully screened and identified 2 clones of HCV NS3 specific ScFv. [