摘要
目的构建腺苷酸激酶4(adenylate kinase 4,AK4)慢病毒过表达质粒,并进行鉴定。方法从Hep G2细胞中扩增AK4基因,将其克隆至载体LV5,构建重组质粒LV5-AK4,经测序鉴定后,将鉴定正确的LV5-AK4和包装质粒p Gag/Pol、p Rev、p VSV-G共转染HEK293T细胞,包装慢病毒;将慢病毒原液进行10倍系列稀释(10^(-1)~10^(-4)),感染HEK293T细胞,检测病毒滴度;慢病毒感染Hep G2细胞,Western blot法检测AK4蛋白的表达水平。结果重组质粒LV5-AK4经测序鉴定证明构建正确,并成功包装成慢病毒,病毒滴度为5×10~8 TU/ml。试验组Hep G2细胞中AK4蛋白的表达水平显著高于空白对照组及阴性对照组(P<0.01)。结论成功构建了携带AK4基因的重组慢病毒过表达质粒,为进一步研究低氧应激细胞中AK4的功能及其分子作用机制奠定了基础。
Objective To construct and identify a lentiviral plasmid for over-expression of adenylate kinase 4（AK4）.Methods AK4 gene was amplified from Hep G2 cells and cloned into LV5 vector. The constructed recombinant plasmid LV5-AK4 was identified by nucleotide sequencing, then co-transfected to HEK293 T cells with packaging plasmids LV5-AK4, p Gag / Pol, p Rev and p VSV-G. The obtained lentivirus was diluted 10-fold serially to the dilutions of 10^（-1）~ 10（-4） and infected to HEK293 T for determination of titer. Hep G2 cells were infected with the lentivirus and determined for expression level of AK4 protein by Western blot. Results Recombinant plasmid LV5-AK4 was constructed correctly as proved by sequencing. The lentivirus was successfully packaged and reached a titer of 5 × 10~8 TU / ml. The expression level of AK4 protein in Hep G2 cells in test group was significantly higher than those in blank and negative control groups（P 〈0. 01）. Conclusion Recombinant lentivirus plasmid with AK4 gene was constructed successfully, which laid a foundation of further study on the function and molecular mechanism of AK4 in hypoxia stress cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第3期248-251,共4页
Chinese Journal of Biologicals
基金
黑龙江八一农垦大学学成
引进人才科研启动计划资助课题(1253HQ013)
黑龙江省教育厅海外学人科研资助项目(1253HQ013)
关键词
腺苷酸激酶4
慢病毒
过表达
Adenylate kinase 4（AK4）
Lentivirus
Over-expression
