Construction and identification of a recombinant lentiviral vector harboring RNAi targeting rat HSP27 gene
- VernacularTitle:大鼠HSP27基因RNA干扰慢病毒载体的构建与鉴定
- Author:
Jie HUANG
;
Liangdi XIE
;
Changsheng XU
;
Huajun WANG
- Publication Type:Journal Article
- Keywords:
HSP27;
RNA interference;
gene therapy;
lentivirus
- From:
Chinese Pharmacological Bulletin
1986;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Aim To construct the recombinant lentiviral RNA interference(RNAi) sequence targeting rat heat shock protein 27(HSP27)gene.Methods Three complementary DNA sequences targeting rat HSP27gene were designed and synthesized.After phosphorylation and annealing,these double strands DNA were cloned into vector pSUPER-basic respectively.Next the ds-oligo DNA which expressed short hairpin RNA(shRNA) was subcloned into lentiviral vector.The transfer plasmid(pNL-HSP27-IRES2-EGFP) was constructed and confirmed by electrophoresis and sequencing.Then the three plasmids of the lentiviral vector: pNL-HSP27-IRES2-EGFP,VSVG,and pHeler were cotransfected to 293T cells by Lipofectamine 2000 to package lentiviral particles.Forty eight hours later the supernatant was collected and the titer of virus was measured by detecting expression leve1 of enhanced green fluorescent protein(EGFP).Results The analysis using restriction endonuclease digestion and sequencing confirmed that the ds-oligo DNA was successfully inserted into the lentiviral vector.The recombinant lentivirus was harvested from 293T cells with a viral titer of 3.12 ?109 fu?L-1.Vascular smooth muscle cells(VSMCs) were infected by lentivirus,and the interfering efficiency was detected by RT-PCR and Western blot.The plasmid with the highest interfering efficiency was pNL-HSP27-IRES2-EGFP-1,and the interfering efficiency was 0.771.Conclusions The recombinant lentivirus containing RNAi targeting HSP27 gene has been successfully constructed,which lay a foundation for further study of the function and gene therapy of HSP27.
