摘要
目的探讨丙型肝炎病毒(HCV)核心蛋白core与其结合蛋白HCBP6在哺乳动物细胞中的相互作用。方法分别扩增HCV核心蛋白core及其结合蛋白HCBP6的cDNA片段,克隆入pGEM-T载体。测序正确后应用双杂交技术把目的片段分别插入哺乳动物细胞双杂交系统的表达质粒pBIND和pACT中构建重组载体。将构建的pBIND-core和pACT-HCBP6重组载体和报告质粒pG5luc共转染HepG2细胞,并设背景对照组(pBIND+pACT)、阳性对照组(pBIND-Myod+pACT-Id)、2个阴性对照组(pBIND-core+pACT、pBIND+pACT-HCBP6)和空白对照组。采用Dual-荧光检测系统和TurnerBiosystemsVeritas微孔板化学发光检测仪检测荧虫素酶活性。结果成功构建重组载体pBIND-core和pACT-HCBP6,共转染HepG2细胞之后,其荧虫素酶活性与海肾素酶活性的比值与各对照组相比有统计学差异(P≤0·05)。结论HCV核心蛋白core与其结合蛋白HCBP6在肝癌细胞系中确有一定的相互作用,为进一步阐明HCV核心蛋白在细胞凋亡及细胞恶变中的作用机制提供了新的思路。
Objective To study the interaction between HCV core protein and HCBP6 in rnammlian cells using CheckMateTM Mammalian Two-Hybrid System. Methods eDNA fragments encoding HCV core protein and HCBP6 were amplified by PCR and subsequently cloned into pGEM-T vector. After verified by sequencing, the target fragments were subcloned into mammalian two-hybrid plasrnids, pBIND and PACT, respectively. The recombinant plasmids, pBIND-core and pACT-HCBP6 were co-transfected into HepG2 cells with reporter plasmid pG51uc, pBIND＋pACT were induced as background controls, pBIND-Myod＋pACT-Id as positive controls, and pBIND- core＋pACT, pBIND＋pACT-HCBP6 as blank controls. The expression of G51uc, which indicated the interaction between HCV core protein and HCBP6 in mammlian HepG2 cells, was assayed through Dual-Luciferase Report Assay System and Turner Biosystems Veritas Microplate Lunimometer. Results The recombinant vectors pBIND-core and pACT-HCBP6 were successfully constructed. When co-transfected into HepG2 cells with reporter plasmid pG51uc, there were significant differences in the luciferase activity in the pBIND-core and pACT-HCBP6 groups compared with every control group. Conclusions HCBP6 can interact with HCV core protein in HepG2 cells, which provides clues for further study on the function of HCBP6 and core proteins, and on the mechanism of HCV core in cell apoptosis and cancer transformation.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第7期790-792,共3页
Medical Journal of Chinese People's Liberation Army
基金
“973”国家重点基础研究发展计划资助项目(2004CB518908)
北京市科委重大科技项目(D08050700650803)
