摘要
目的根据丙型肝炎病毒（HCV）丝氨酸蛋白酶4个多肽底物切点的氮基酸序列特异性，设计1个多位点的竞争性抑制多肽序列。方法采用中心模板法PCR合成多肽基因；利用原核高效表达载体pBVIL1在大肠杆菌HB101中。表达目的多肽；提取包涵体，用离子交换层析纯化蛋白；在体外添加到蛋白酶和NSSA—B片段构建的反应系统中。用SDS—PAGE鉴定多肽对病毒蛋白酶的抑制活性。结果正确合成了多肽基因，重组载体pBVIL1／IP在HB101中表达了目的多肽；纯化后，获得电泳纯多肽；体外初步测定证实，随多肽浓度增加，蛋白酶底物降解受到抑制。结论原核表达的重组抑制多肽，体外对HCV蛋白酶活性具有抑制作用。
Objective To synthesize an inhibitory peptide against serine protease of hepatitis C virus （HCV） and explore its inhibition ability in vitro. Methods Based on the sequence characteristics of four natural substrates of protease in HCV, the gene sequence of an inhibitory peptide against HCV serine protease was designed and directly synthesized by PCR. The segment was subcloned into prokaryotic expression vector pBVIL1, resulting in the construction of the recombinant plasmid pBVIL1/IP, which was then transformed into E. coli HB101 strain. Purified by ion exchange chromatography, the expressed protein was added into an in vitro system, which was comprised of the inhibitory peptide （ expressed protein）, protease and the substrate, i.e. a NSSA-B fragment, in phosphate buffer. Then SDS-PAGE was performed to test the inhibition effect of the polypeptide. Results The recombinant expression vector pBVIL1/IP containing target gene was successfully constructed. The peptide expressed as inclusion body was identified by SDSPAGE. The degradation of protease substrate NSSA-B fragment is inhibited proportionally with the increasing concentrations of the peptide. Conclusion The recombinant peptide shows inhibitory effect on HCV protease in vitro.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2008年第2期119-122,共4页
Chinese Journal of Microbiology and Immunology
