利用毕赤酵母系统表达具有催化活性的丙型肝炎病毒 (HCV)NS3蛋白酶 .将PCR直接扩增的病毒NS3丝氨酸蛋白酶基因和重组的带有辅酶的单链NS3 NS4A蛋白酶基因 ,分别插入表达载体pPICZαA的EcoRⅠ和XbaⅠ克隆位点 ,转化毕赤酵母GS115 ,可溶性表达NS3蛋白酶和单链NS3 4A蛋白酶 ;ELISA法测定表达蛋白酶的抗原性 ;原核高效表达载体pBVIL1表达酶切底物NS5A B片段 ,体外与蛋白酶共同温育 ,SDS PAGE鉴定蛋白酶催化活性 .载体测序结果表明 ,重组载体pPICZαA NS3和pPICZαA NS3 4A中的目的基因序列插入正确 ;SDS PAGE结果显示 ,培养物上清中存在分泌型目的蛋白带 ;ELISA结果证实 ,表达蛋白与HCV阳性血清有结合活性 ;蛋白酶与底物NS5A B片段不同作用时间的SDS PAGE ,看到约 2 4kD处底物条带的分解 .说明用毕赤酵母表达系统成功地表达了可溶性HCVNS3和单链NS3 4A蛋白酶 ;两种结构形式的蛋白酶在体外系统中都有催化活性 ,同时也都具有抗原性 .该研究为大量和方便地获得有催化活性的HCVNS3蛋白酶提供了有效途径 .
Abstract
Non-structural protein 3(NS3) of hepatitis C virus (HCV),the serine protease, is a key functional protein, which is responsible for the processing of HCV polyprotein. NS3 protease gene and single chain NS4A-NS3(scNS3/4A) conjugated protease gene were subcloned into pPICZαA vector,and then the recombinant plasmids were transfected into Pichia pastoris GS115. The two types of recombinant viral proteases were secretively expressed in yeast. In order to evaluate the catalytic activity of the enzymes in vitro, an NS5A-B fragment containing a cleavage site of the protease was subcloned into pBVIL1 vector and highly expressed in E coli. As a result, NS3 and scNS3/4A proteases were proved to be specific antigenicity detected by ELISA and to be able to degrade the substrate NS5A-B in a system in vitro with SDS-PAGE assay. Both of proteases can further be used to study the catalytic activity and find the inhibitors against this protease.
关键词
丙型肝炎病毒 /
丝氨酸蛋白酶 /
毕赤酵母 /
可溶性表达
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Key words
HCV /
serine protease /
Pichia pastoris /
soluble expression
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