摘要
探讨丙型肝炎病毒(HCV)F基因对PI3K表达的影响。应用PCR技术从含有HCV全长开放阅读框的质粒中获得HCV-F基因片段,利用基因重组技术将其克隆至真核表达载体pcDNA3.0(-)中。通过酶切、PCR及测序鉴定,HCV-F基因已正确插入到pcDNA3.0(-)中,再利用脂质体转染HepG2细胞。经RT-PCR及Westernblot检测,HCV的F基因在HepG2细胞中获得表达,而且在表达重组HCV-F的转染HepG2细胞中,检测到PI3K蛋白的表达。实验表明HCV-F可在体外激活PI3K及其信号通路。
To reveal the influence of HCV-F on PI3K, The relationship between F and PI3K in vitro was explored. Full length F gene of HCV was amplified by PCR, using the plasmid containing HCV fulllength open reading frame （ORF） as template, and cloned into the eukaryotic expressing plasmid pcDNA3.0（-） by DNA recombination technique. The recombinant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by direct sequencing. Then both the recombinant vector pcDNA3. 0-F and the control vector pcDNA3. 0 （-） were transfected HepG2 cell using Lipofectamin2000. Expressing of F gene was proved by RT-PCR and Western blot analysis in transfected HepG2 cell. The PI3K protein expressed in the HepG2 cell expressing recombinant HCV-F was further examined. HCV-F can activate PI3K in vitro and its signal cascade pathway.
出处
《药物生物技术》
CAS
CSCD
2008年第4期235-238,共4页
Pharmaceutical Biotechnology
基金
江苏省自然科学基金资助(BK2004011)
