Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene
10.3785/j.issn.1008-9292.2014.03.013
- VernacularTitle:整合素连接激酶基因敲降和黑色素瘤分化相关基因过表达慢病毒载体构建和鉴定
- Author:
You-Ping YANG
1
;
Yan DING
;
Ji-Rong WANG
;
Ling-Hui ZENG
;
Hong-Xia LIN
;
Yang-Li ZHU
;
Hong-Wei WU
;
Ruo-Yan WANG
;
Jian-Min ZHANG
;
Rong-Biao YING
Author Information
1. 浙江省台州市肿瘤医院
- Keywords:
Integrins;
Phosphotransferases;
Prostatic neoplasms;
Interleukins;
Genes,tumor suppressor;
Lentivirus/genetics;
Genetic vectors;
Gene expression
- From:
Journal of Zhejiang University. Medical sciences
2014;(2):193-199
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene.Methods:Based on the human ILK gene sequences , RNAi target sequences were designed and cloned into the lentiviral vector pSicoR -eGFP by restriction endonuclease HpaⅠand XhoⅠdouble digestion and T 4 DNA ligase ligation . Based on the human mda7 gene sequences , PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro.After the candidate clones were identified by DNA sequencing , the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles .Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector .The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot , respectively .The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively.Results: ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed .Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector .The transfection efficiency of the collected virus exceeded 90%in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency .The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly .The mda7-pLVX-Puro lentiviral vector increased the expression of mda 7 in PC-3 cells, and the ability was maintained for one month.Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells ( Ps <0.05 ) . Conclusion: The lentiviral vectors of ILK knockdown and mda 7 over-expression have been successfully constructed and identified . The recombinant lentivirus can efficiently infect human prostate cancer PC -3 cells, in which ILK expression is inhibited and mda 7 is over-expressed .
