聚合酶链反应诱导丙型肝炎病毒(HCV)蛋白酶活性位点的突变及突变HCVNS3/4a蛋白的表达

目的 以聚合酶链反应（PCR）突变方法诱导丙型肝炎病毒（HCV）蛋白酶活性位点ser1165的突变，获得全长非结构基因3（NS3）／4a的表达与纯化。方法 分别以NS3 N端正向引物与诱变反向引物，诱变正向引物与NS4a C端反向引物获得2个PCR产物，产物纯化后在新的PCR反应体系中加入以上2个PCR产物与NS3 N端正向引物、NS4a C端反向引物。再次PCR扩增突变模板，分别与野生型模板重组入表达载体pET26-Ub，转化大肠杆菌BL21（DE3）pCG1，诱导表达后经菌体裂解、纯清化、硫酸铵沉淀、DEAE－Sepharose、NTA纯化，Western blot分析表达蛋白的特异性及PCR诱导突变使HCV蛋白酶活性位点失活的作用。结果 获得诱导突变的模板，Western blot证实该突变可完全阻断对NS3丝氨酸蛋白酶与NS3螺旋酶间的切割，部分阻断了螺旋酶与NS4a间的切割，纯化后的HCV NS3／4a蛋白在SDS－PAGE胶上显示为双带。结论 PCR突变方法简便、有效，获得丝氨酸蛋白酶失活的NS3蛋白表达，NS3蛋白与NS4a蛋白以复合物形式存在。

Inactivation of hepatitis C virus (HCV) nonstructural (NS) 3 serine protease by PCR-mutagenesis and expression of mutant HCV NS3/4a

Objective To introduce a ser1165-leu1165 substitution into the sequence of the HCV NS3 protein and obtain purified entire HCV NS3/4a protein. Methods Two DNA fragments were amplified by PCR, the first with the sense primer corresponding to N-terminal of NS3 and an antisense primer 4, and the second with a sense primer 3 and an antisense primer corresponding to C-terminal of HCV NS4a. Primer 3 and 4 contained the nucleotide change converting the TCG codon of serine into TTG codon of leucine. Mutant PCR product and wild PCR product were recombinanted into pET26-Ub and transformed into BL21(DE3)pCG1, respectively. After induction and expression, the strain was broken and the proteins were purified by clarification, AmSO 4 cut, DEAE-Sepharose and NTA resin. Specific proteins and inactive HCV serine protease were confirmed in Western blot. Results Mutant template was obtained by PCR-mutagenesis. Western blot indicated that mutation in ser1165 abolished internal cleavage of the NS3 protein completely and only inactivated part protease activity at NS3/4a junction. Purified NS3/4a protein was double bands on SDS-PAGE. Conclusions PCR-mutagenesis is simple and effective. Entire NS3/4a protein with inactive serine proteinase activity was obtained and NS3 protein was obtained as the NS3/NS4a noncovalent complex.