可溶性HCV非结构蛋白NS3人源单链可变区抗体在大肠杆菌中的表达

目的 获得可溶性的抗丙型肝炎病毒(HCV)非结构蛋白NS3的人源单链可变区抗体(ScFv),为得到纯度高、活力强的NS3 ScFv和进一步HCV的治疗奠定基础。方法 采用噬菌体表面展示技术,以重组的HCV非结构蛋白NS3为包被抗原,从噬菌体单链可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性较强的HCVNS3人单链抗体ScFv片段克隆,并对其进行DNA序列及免疫活性测定。从噬菌体抗体阳性克隆中提取质粒转化琥珀突变非抑制型大肠杆菌HB2151;IPTG诱导表达HCV NS3可溶性单链抗体;ELISA和dot blot检测其抗原结合特异性。结果 克隆了HCV NS3的单链可变区抗体基因,经DNA酶切和序列分析表明,该抗体基因由747个碱基组成。ELISA和dot blot结果表明,在大肠杆菌HB2151中经IPTG诱导表达的可溶性HCV NS3的单链可变区抗体,具有结合丙型肝炎病毒非结构蛋白NS3的特异性和免疫活性。结论 克隆、鉴定并在大肠杆菌HB2151中表达了可溶性的ScFv-NS3。

Expression of soluble human single chain Fv antibody to hepatitis C NS3 antigen in E. coli

Objective Expression of human single chain Fv antibody to hepatitis C virus NS3 antigen in E. coli. Methods The recombinant phages were panned by NS3 antigen which was coated in a microtiter plate, after five rounds of biopanning, 66 clones were identified specific to NS3 antigen. E. coli HB2151 was transformed and induced by IPTG. The specificity of ScFv was evaluated by ELISA and dot blot hybridization. Results ScFv-NS3 DNA digestion and sequence data showed that the ScFv gene is composed of 747bp. ELISA and dot blot demonstrated that the soluble human single chain Fv antibody to hepatitis C virus NS3 antigen has a specific combination character with hepatitis C virus NS3 antigen. Conclusion Identification and expression of HCV NS3 specific ScFv from E. coli were successfully achieved.