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目的在大肠杆菌ＸＬ_１－Ｂｌｕｅ中表达可溶性的抗ＨＣＶ非结构蛋白ＮＳ_３的人源单链可变区抗体（ｓｉｎｇｌｅ－ｃｈａｉｎ ｖａｒｉａｂｌｅ ｆｒａｇｍｅｎｔ ａｎｔｉｂｏｄｙ，ＳＣＦＶ）。方法以重组的 ＨＣＶ ＮＳ_３为抗原，利用噬菌体抗体库技术筛选含有抗－ＨＣＶ ＮＳ_３ ＳＣＦｖ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ＸＬ_１－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 ＨＣＶ ＮＳ_３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ＨＣＶ ＮＳ_３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 ＸＬＩ-Ｂｉｌｌ６中表达的 ＨＣＶ ＮＳ_３的单链抗体，可与不同来源的ＮＳ_３抗原结合。结论大肠杆菌ＸＬ_Ｉ－Ｂｌｕｅ表达的ＮＳ_３－ＳＣＦＶ具有结合不同来源的ＨＣＶＮＳ３的活性和特异性。
Objective To express soluble single-chain variable fragment antibody (SCFV) against nonstructural protein NS_3 in E.coli XL_1-Blue. Methods The recombinant HCV NS_3 was used as antigen to screen phage clones containing anti-HCV NS_3 SCFv gene using phage antibody library technology. Plasmids were extracted from phage antibody-positive clones and identified by SfiI / NotI digestion. The plasmids were subcloned into pCANTAB5E vector and transformed into E.coli XL_1-Blue. The plasmids were extracted for DNA sequencing. Isopropyl thio-β-D- Glycosides (isopropylthio-β-D-galactoside, IPTG) induced the expression of HCV NS3 soluble single chain variable region antibody. ELISA and blot blot hybridization to detect their binding activity to different origins of the antigen. Results The scFv gene of HCV NS_3 was confirmed by restriction enzyme digestion and sequence analysis. The result showed that the antibody gene consisted of 750bp. The results of ELISA and dot blot hybridization showed that the gene expressed in E. coli XLI-Bill6 The single chain antibody of HCV NS3 can bind NS3 antigen of different origin. Conclusion The NS_3-SCFV expressed by E. coli XL_I-Blue has the activity and specificity of binding HCVNS3 from different sources.