柯萨奇B4病毒非结构蛋白P_2C基因的克隆与表达  

Cloning and Expression of Gene Encoding Nonstructural Protein P_2C of Coxsackievirus B4

作　　者：张宸豪[1,2] 任旷[2] 马爱新[2] 方芳[1] 

机构地区：[1]吉林大学基础医学院免疫学教研室,长春130021 [2]吉林医药学院病原学教研室,吉林132003

出　　处：《中国生物制品学杂志》2009年第3期246-248,255,共4页Chinese Journal of Biologicals

摘　　要：目的克隆并表达柯萨奇B4病毒(CVB4)非结构蛋白P2C基因。方法提取CVB4总RNA,RT-PCR扩增P2C基因,克隆入pUCm-T载体中,进行酶切和测序鉴定。双酶切重组质粒pUCm-T-P2C,将P2C基因片段定向亚克隆至原核表达载体pMAL-C2中,转化大肠杆菌JM109,IPTG诱导表达,SDS-PAGE和Western blot鉴定表达产物。结果RT-PCR扩增得到987bp的基因片段,测序结果与GenBank公布的P2C基因序列一致。pMAL-C2-P2C经双酶切鉴定,证明构建正确。表达的融合蛋白相对分子质量约78000,表达量约占菌体总蛋白的25%,且具有良好的反应原性。结论已成功克隆并表达了CVB4P2C基因,为进一步研究其生物学活性奠定了基础。Objective To clone and express the gene encoding nonstructural protein P2C of Coxsackievirns B4 （CVB4）. Methods The total RNA of CVB4 was extracted for amplification of P2C gene by RT-PCR. The amplified P2C gene was cloned into vector pUCm-T, and the constructed recombinant plasmid pUCm-T-P2C was identified by restriction analysis and sequencing then digested with BamH I and Hind m. The obtained P2C gene fragment was subcloned into prokaryotie expression vector pMAL-C2, and the constructed recombinant plasmid pMAL-C2-P2C was transformed to E. coli JMI09 for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The gene fragment at a length of 987 bp was amplified by RT-PCR, with identical sequence to that of P2C gene reported in GenBank. Restriction analysis proved that recombinant plasmid pMAL-C2-P2C was constructed correctly. The expressed fusion protein, with a relative molecular mass of about 78 000, contained about 25% of total somatic protein and showed good reaetogenicity. Conclusion The P2C gene of CVB4 was successfully cloned and expressed, which laid a foundation of further study on biological activity of P2C protein.

关 键 词：柯萨奇B4病毒 非结构蛋白 P2C基因 克隆 表达 

分 类 号：Q785[生物学—分子生物学] Q786