HCV丝氨酸蛋白酶小分子底物构建及GST融合表达

Construction of a Small Peptide as a Surrogate of Natural Substrate of HCV Serine Protease and Fusion Expression with GST in Prokaryotic Cells

丝氨酸蛋白酶是丙型肝炎病毒重要的功能蛋白和药物作用靶点,其通过分子内(cis)和分子间(trans)方式催化水解前体蛋白,释放病毒功能蛋白。目的:为深入研究病毒蛋白酶活性和抑制剂鉴定需要,实验研究参照丙型肝炎病毒1a亚型菌株蛋白酶天然底物的氨基酸序列特点,设计了一段包含两个天然底物酶切位点的小分子多肽2S,并进行了原核表达。方法:利用PCR方法,合成2S小分子多肽基因,目的基因两端引入BamHI和EcoRI两个限制性酶切位点,双酶切后将基因与表达载体pGEX-4T-2重组,转化大肠杆菌DH5α,经化学诱导进行GST融合蛋白表达,通过亲和层析柱纯化目的蛋白。纯化的GST-2S融合蛋白在体外反应系统进行酶切鉴定,SDS-PAGE和ELISA鉴定酶切结果。结果:PCR合成的小分子底物多肽2S基因,经与表达载体重组后测序,证实基因序列正确。采用0.5mmol/L浓度的IPTG诱导工程菌过夜,获得表达的目的蛋白,经分离纯化得到融合蛋白GST-2S。GST-2S在体外磷酸盐缓冲系统中与丝氨酸蛋白酶反应,15%SDS-PAGE鉴定酶切产物,证实融合蛋白底物条带明显消失,ELISA结果同样说明融合蛋白的底物活性。结论:含有两个天然底物酶切位点的小分子多肽可以替代病毒天然底物,实验结果为丙型肝炎病毒丝氨酸蛋白酶活性研究和酶抑制剂研究奠定了方法学基础。

Serine protease of hepatitis C virus,which is a key viral non-structural protein and anti-virus target,could degrade HCV polyprotein with cis and trans way to release viral function proteins.Objective：To develop a method for evaluating the activities of serine protein or its inhibitors in vitro,a small recombinant peptide（ named 2S） as a surrogate of natural substrate of HCV serine protease was designed and its specific sequence was refered to subtype 1a containing amino acid sequences of two linkage sites between HCV NS3 and NS4A,and NS4A and NS4B.A small peptide 2S was expressed finally in prokaryotic cells with GST fusion.Methods：2S gene was synthesized with PCR technique while bath BamH I and EcoR I restriction sites were inserted into 5＇ and 3＇ ends of the gene.The desired gene was enzymatically performed with BamH I and EcoR I and subcloned into pGEX-4T-2 vector.The recombinant plasmid pGEX-4T-2s was identified with sequencing after transformed into competent cells DH5α.The engineering bacteria was induced overnight by 0.5mmol/L IPTG and GST-2S fusion protein was purified with GST affinity column and applied to be degraded with raw protease in phosphate buffer system in vitro.In addition,the catalytic characteristic was analyzed through SDS-PAGE electrophoresis and ELISA.Result：The recombinant plasmid pGEX-4T-2s was successfully constructed and the desired protein purified was obtained by affinity column.The detection in PB catalytic system showed that GST-2S belt clearly disappeared with SDS-PAGE and A450 value of the test panel was much lower than positive control with ELISA.Conclusion：A small recombinant peptide designed with two cutting sites of HCV serine protease could be degraded by the enzyme in an in vitro system and could be a surrogate as natural substrate HCV serine protease.