丙型肝炎病毒NS3蛋白人源基因工程单链抗体的表达

目的在大肠杆菌XL1-Blue中表达可溶性的抗HCV非结构蛋白NS3的人源单链可变区抗体（single-chainvariablefragmentantibody，ScFv）。方法以重组的HCVNS3为抗原，利用噬菌体抗体库技术筛选含有抗-HCVNS3ScFv基因的噬菌体克隆。从噬菌体抗体阳性克隆中提取质粒，经SfiI/NotⅠ酶切鉴定后，亚克隆到pCANTAB5E载体；转化大肠杆菌XL1-Blue，提取质粒进行DNA序列测定；异丙基硫代-β-D-半乳糖苷（isopropylthio-β-D-galactoside，IPTG）诱导表达HCVNS3可溶性单链可变区抗体。ELISA和斑点吸印杂交检测其与不同来源的抗原的结合活性。结果筛选到的HCVNS3的单链抗体基因，经限制性内切酶酶切和序列分析表明，该抗体基因由750bp组成，ELISA和斑点吸印杂交结果表明，在大肠杆菌XL1-Blue中表达的HCVNS3的单链抗体，可与不同来源的NS3抗原结合。结论大肠杆菌XL1-Blue表达的NS3-ScFv具有结合不同来源的HCVNS3的活性和特异性。

Expression of human single-chain variable fragment antibody against non-structural protein 3 of hepatitis C virus antigen in E coli.

Objective To express human single-chain variable fragment (ScFv) antibody against non-structural protein 3 (NS_3) of hepatitis C virus in E. colt. Methods The recombinant phages were panned by NS_3 antigen which was coated in a microtiter plate. After five rounds of biopanning, 66 clones were identified specific to NS_3 antigen. E. colt host XL_1-Blue was transformed and induced by IPTG. The specificity of ScFv was evaluated by ELISA and dot blot hybridization. Results ScFv-NS_3 DNA digestion and sequencing data showed that foe ScFv gene was composed of 750 hp. ELISA and dot blot hybridization demonstrated that the soluble human single- chain Fv antibody to hepatitis C virus NS3 antigen could combine different origins of NS_3 antigen. Conclusion NS_3-ScFv antibody expressed by E. colt host XL_1- Blue has the activity and specificity to combine different origins of HCV NS_3 antigen.