Construction of recombinant lentiviral vector of si RNA for transforming growth factor-beta typeⅠ receptor gene and its expression in human embryonic lung fibroblasts
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Construction of recombinant lentiviral vector of si RNA for transforming growth factor-beta typeⅠ receptor gene and its expression in human embryonic lung fibroblasts
Journal of Chongqing Medical UniversityVol. 41, Issue 9, Pages: 915-922(2016)
Lin Shihui, Fu Juan, Wang Chuanjiang, et al. Construction of recombinant lentiviral vector of si RNA for transforming growth factor-beta typeⅠ receptor gene and its expression in human embryonic lung fibroblasts. [J]. Journal of Chongqing Medical University 41(9):915-922(2016)
Lin Shihui, Fu Juan, Wang Chuanjiang, et al. Construction of recombinant lentiviral vector of si RNA for transforming growth factor-beta typeⅠ receptor gene and its expression in human embryonic lung fibroblasts. [J]. Journal of Chongqing Medical University 41(9):915-922(2016) DOI: 10.13406/j.cnki.cyxb.001023.
Construction of recombinant lentiviral vector of si RNA for transforming growth factor-beta typeⅠ receptor gene and its expression in human embryonic lung fibroblasts
Objective:To construct lentiviral vector for RNA interference(RNAi)of the transforming growth factor beta receptor Ⅰ(TGFβRⅠ)gene and to detect its silencing efficiency in the human embryonic lung fibroblasts MRC-5. Methods:Four kinds of si RNA lentiviral vectors targeting to TGFβRⅠgene were constructed
and the vectors were identified by PCR and DNA sequencing. First the effective recombinant plasmids and the helper plasmids were mixed together
and then the mixture of plasmids was transfected into293 T cells. At last
the viral titers was measured. The human embryonic lung fibroblasts cell line MRC-5 infected by four recombinant lentivirus vectors
respectively. Then
q RT-PCR and Western blot were used to detect the expression of TGFβRⅠm RNA and protein.Results:Double restriction digestion analysis and DNA sequencing showed that the sh RNA sequences was successfully inserted into the lentiviral vectors. The titer of RNAi virus group 1-4 was 6.37×107TU/ml
1.65 ×108 TU/ml
4.50 ×108 TU/ml and 2.31 ×108TU / ml
respectively. The titer of negative control group was1.79×109TU/ml and the efficiency of infecting virus of MRC-5 was approximately 93%. The expression of TGFβRⅠm RNA was significantly lower in si RNA-1-4 groups than in normal control group and negative control group(P =0.000). And
the si RNA- 2 had the highest silencing efficiency. The result of Western blot analysis showed that the expression of TGFβRⅠprotein was significantly decreased in the si RNA-1-4 groups compared with that of normal group(P=0.000)
and the si RNA-2 had the greatest reduction. Conclusion :The optimal si RNA recombi nant lentiviral vector targeting to TGFβRⅠ gene is successfully screened and constructed