摘要
目的:构建重组慢病毒质粒pSin-EF2-Puro-DAB2IP并转染前列腺癌PC3细胞,观察其转染效率及其在PC3细胞中的表达。方法:以人前列腺癌PC3细胞基因组cDNA为模板PCR扩增获得目的基因DAB2IP片段后,应用基因重组技术将目的片段克隆到PC3-pSin慢病毒表达载体,经PCR、双酶切和测序鉴定后,重组慢病毒表达质粒和包装质粒共转染293FT细胞,获得携带DAB2IP基因的重组慢病毒。取病毒上清感染人前列腺癌PC3细胞,以PC3-pSin-EF2为对照,分别采用RT-QPCR、Western blotting法检测DAB2IP在靶细胞中的表达水平。结果:重组慢病毒质粒pSin-EF2-Puro-DAB2IP经PCR、EcoRⅠ和NheⅠ双酶切鉴定结果与目的基因条带吻合,克隆测序结果与NCBI收录的DAB2IP基因序列(NM_138709)完全一致。重组慢病毒质粒转染293FT细胞收获慢病毒上清可高效感染PC3细胞,对照组细胞株PC3-pSin-EF2及过表达细胞株PC3-pSin-EF2-DAB2IP内DAB2IP基因的相对拷贝数分别为0.001±0.000和0.158±0.013,与对照组细胞比较,过表达细胞内DAB2IP基因mRNA表达水平上调(179.37±15.89)倍,差异有统计学意义(P<0.001);Western blotting法,对照组PC3-pSin-EF2细胞中DAB2IP蛋白表达水平为内参的(1.002±0.783)倍,PC3-pSin-EF2-DAB2IP细胞组为内参的(2.431±0.892)倍,过表达组DAB2IP蛋白表达水平为对照组的(2.415±0.961)倍,差异有统计学意义(P<0.01)。结论:成功构建携带DAB2IP基因的慢病毒表达载体pSin-EF2-Puro-DAB2IP,并使目的基因在靶细胞中稳定表达,为进一步研究DAB2IP基因的相关功能提供了优质的稳定转染载体。
Objective To construct recombinant lentiviral vector pSin-EF2-Puro-DAB2IP transfect prostate carcinoma PC3 cells,and to observe its transfection rate and expression level in PC3 cells.Methods The cDNA sequences specifically targeting the DAB2IP gene were designed and cloned into lentiviral vector PC3-pSin using DNA recombinant technique.Using PC3-pSin-EF2 as control,the 293T cells were transfected by Lipofectamine reagent for lentiviral particles packaged, and viral titer was determined. The DAB2IP mRNA and protein expression levels were examined by RT-PCR and Western blotting methods. Results The PCR and sequencing analysis results confirmed that the DAB2IP gene sequence was consistent with the sequence in GeneBank. The number of DAB2IP gene copy in PC3-pSin-EF2-DAB2IP cells and its control PC3-pSin-EF2 cells were 0.001±0.000 and 0.158 ± 0.013,respectively.Compared with control cells,the overexpression of mRNA in PC3-pSin-EF2-DAB2IP cells upregulated （179.370 ± 15.891）times,the difference was statistically significant （P〈0.001）. Compared with internal reference and control cells, the expression levels of DAB2IP protein in PC3-pSin-EF2-DAB2IP cells upregulated （2.431 ± 0.892）times and （2.415 ± 0.961）times respectively,the differences were statistically significant （P〈0.001）.Conclusion The lentiviral vector of the DAB2IP gene pSin-EF2-Puro-DAB2IP is successfully constructed,and its targeted gene is stably expressed in the targeted cells,which provides a basis for the further functional study of DAB2IP.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2014年第5期938-942,共5页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金面上项目资助课题(81272849)
广东省科技厅科技计划项目资助课题(2011B061200021)
