[Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene].
To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene.



Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 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.



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 mda7 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).



The lentiviral vectors of ILK knockdown and mda7 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 mda7 is over-expressed.
Taizhou Cancer Hospital, Wenling 310075, China.
You-ping
YP
Yang
Taizhou Central Hospital, Taizhou 318000, China.
Yan
Y
Ding
Zhejiang University City College, School of Medicine, Hangzhou 310015, China.
Ji-rong
JR
Wang
Zhejiang University City College, School of Medicine, Hangzhou 310015, China.
Ling-hui
LH
Zeng
Taizhou Cancer Hospital, Wenling 310075, China.
Hong-xia
HX
Lin
Taizhou Cancer Hospital, Wenling 310075, China.
Yang-li
YL
Zhu
Taizhou Cancer Hospital, Wenling 310075, China.
Hong-wei
HW
Wu
Taizhou Cancer Hospital, Wenling 310075, China.
Ruo-yan
RY
Wang
Taizhou Cancer Hospital, Wenling 310075, China.
Jian-min
JM
Zhang
Taizhou Cancer Hospital, Wenling 310075, China.
Rong-biao
RB
Ying
D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D007378:Interleukins / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D000099200:Scaffold Protein ILK; D000099180:Interleukin-24
D007378:Interleukins; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D000099200:Scaffold Protein ILK; D000099180:Interleukin-24