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目的利用原核表达载体pBVIL1,表达丙型肝炎病毒非结构蛋白3(NS3)丝氨酸蛋白酶催化底物NS5A-B小分子,为进一步研究蛋白酶活性提供方法学。方法将PCR合成的NS5A-B(2412-2427aa)基因直接插入高效原核表达载体pBVIL1,融合表达NS5A-B片段,包涵体形式的表达产物用8mol/L脲溶解后,采用离子交换和凝胶过滤两步纯化。利用SDS-PAGE、Western blot和ELISA方法,于37℃酶催化条件下,分析NS5A-B底物的降解活性。结果(1)构建了pBVIL1/NS5A-B重组质粒,鉴定证明NS5A-B基因片段正确地插入表达载体上。融合蛋白在转化质粒的HB101菌中得到高效表达。分离纯化重组蛋白后浓度可达0.73g/L。(2)在NS3蛋白酶作用不同时间后,用SDS-PAGE和Western blot证实,底物带可被明显降解,ELISA分析也证明,NS5A-B具有酶底物活性。结论含有酶切位点的NS5A-B融合蛋白可被NS3丝氨酸蛋白酶有效地降解,可用作为NS3蛋白酶的底物,可替代全长NS5A-B和化学合成肽用于酶活性和酶阻断剂的研究。
Objective To express a small molecule of NS5A-B catalyzed by serine protease of nonstructural protein 3 (NS3) of hepatitis C virus by using prokaryotic expression vector pBVIL1, providing a methodology for further study of protease activity. Methods The NS5A-B (2412-2427aa) gene was inserted into pBVIL1 vector and fused with NS5A-B fragment. The expressed product of inclusion body was dissolved in 8mol / L urea and purified by ion exchange and gel filtration Two-step purification. The degrading activity of NS5A-B substrate was analyzed by SDS-PAGE, Western blot and ELISA at 37 ℃. Results (1) The recombinant plasmid pBVIL1 / NS5A-B was constructed and verified that the NS5A-B gene fragment was correctly inserted into the expression vector. The fusion protein was highly expressed in the HB101 transformed plasmid. After isolation and purification of recombinant protein concentration of up to 0.73g / L. (2) Substrate bands could be significantly degraded by SDS-PAGE and Western blot at different time after NS3 protease action. ELISA assay also showed that NS5A-B had enzyme substrate activity. Conclusion The NS5A-B fusion protein containing the cleavage site can be effectively degraded by NS3 serine protease and can be used as a substrate for NS3 protease to replace the full-length NS5A-B and chemically synthesized peptide for enzyme activity and enzyme blockers the study.