Construction of a lentivirus vector for Trail gene in rats
10.3969/j.issn.2095-4344.2014.02.017
- VernacularTitle:构建含肿瘤坏死因子相关凋亡诱导配体基因慢病毒载体
- Author:
Hai ZHANG
;
Zhengfang JIANG
;
Yang LIU
;
Bo ZHANG
;
Guiqiang WU
;
Lingyong ZENG
- Publication Type:Journal Article
- Keywords:
TNF-related apoptosis-inducing ligand;
genes;
plasmids;
polymerase chain reaction;
blotting;
western
- From:
Chinese Journal of Tissue Engineering Research
2014;(2):265-270
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Adenovirus, expressing within a limited period, can limit the expression time and amount of target genes that is not conducive to ongoing experiments. Here, we select adenovirus as vectors for genetic recombination with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Trail) gene fragment.
OBJECTIVE:To explore the construction of recombinant lentiviral vector carrying Trail in rats
METHODS:The Trail gene was obtained:according to GenBank in rat Trail gene sequence (NM_145681.1), we designed the gene specific primers of Trail-Age I-F and Trail-AgeI-R, and used AgeI as enzyme cutting site. PCR was applied to amplify Trail gene from rat cDNA Library and construct recombinant plasmids after cutting Trail gene to be cloned into expression vector GV218 by AgeI. Recombinant plasmids were transfected into 293T cells by Lipofeetamine2000 encapsulated recombinant plasmid and auxiliary packaging carrier. The Trail protein of lentiviral plasmids was expressed. Fol owing virus col ection, we identified virus titer and extracted protein from cells to detect Trail expression by western blot assay.
RESULTS AND CONCLUSION:Screened positive Escherichia coli DH5a competent cells were sequenced with 861 bp, which was consistent with Trail nucleotide sequence in GenBank. After transfection 2 days, virus liquid was col ected and confirmed as recombinant plasmid including Trail gene by PCR and Trail proteins expressed in 293T cells by western blot assay. Hole dilution method and real-time fluorescent quantitative PCR determination showed that the virus titer was 2×109 TU/mL. In this study, recombinant lentiviral vector carrying Trail is successful y constructed by homologous recombination in Escherichia coli.
