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目的:构建转化生长因子β型受体Ⅰ(transforming growth factor beta receptorⅠ,TGFβRⅠ)基因RNA干扰(RNA interference,RNAi)慢病毒载体并在人胚肺成纤维细胞株MRC-5上鉴定其沉默效应。方法:构建4种针对人TGFβRⅠ基因不同干扰靶点的慢病毒干扰载体,PCR筛选阳性克隆,测序鉴定,将筛选出的4种有效重组慢病毒干扰载体和其它辅助质粒一起共转染293T细胞并测定病毒滴度。最后将4种重组的慢病毒干扰载体分别感染MRC-5细胞,采用Real-time PCR和Western blot检测它们对靶基因的沉默效率。结果:经双酶切鉴定和DNA测序,证实短发夹RNA正确插入慢病毒载体且插入的序列正确。4种重组慢病毒干扰载体经293T细胞成功包装后,其病毒滴度分别为:si RNA-1 6.37×107 TU/ml、si RNA-2 1.65×108 TU/ml、si RNA-3 4.50×108TU/ml、si RNA-4 2.31×108TU/ml,阴性对照组载体包装后的病毒滴度为:1.79×109 TU/ml。4种重组慢病毒干扰载体感染MRC-5细胞的效率均为93%左右。与正常对照组和阴性对照组比较,在感染4种重组慢病毒干扰载体(si RNA-1~4)后,MRC-5细胞内TGFβRⅠm RNA表达水平均明显下降(P=0.000),其中TGFβRⅠ-si RNA-2下降最明显,沉默效率最好。Western blot结果与q RT-PCR结果一致,4种重组慢病毒干扰载体均能使MRC-5细胞内TGFβRⅠ蛋白表达明显下降(P=0.000),且TGFβRⅠ-si RNA-2干扰效率最大。结论:筛选并构建了能有效沉默TGFβRⅠ基因的最佳RNAi重组慢病毒载体——TGFβR-si RNA-2,从而为后续研究奠定基础。
OBJECTIVE: To construct the RNA interference (RNAi) lentiviral vector of transforming growth factor beta receptor Ⅰ (TGFβRⅠ) gene and identify its silencing effect on human embryonic lung fibroblast cell line MRC-5. METHODS: Four lentiviral vectors targeting human TGFβRⅠ gene were constructed. The positive clones were screened by PCR and sequenced. The four recombinant lentivirus vectors screened were co-transfected into 293T cells with other helper plasmids. Determination of virus titer. Finally, four recombinant lentiviral vectors were used to infect MRC-5 cells, respectively. Real-time PCR and Western blot were used to detect the silencing efficiency of target genes. Results: Double-restriction enzyme digestion and DNA sequencing confirmed that the short hairpin RNA was correctly inserted into the lentiviral vector and the inserted sequence was correct. The viral titers of the 4 kinds of recombinant lentiviral vector after being successfully packaged by 293T cells were: si RNA-1 6.37 × 107 TU / ml, si RNA-2 1.65 × 108 TU / ml, si RNA-3 4.50 × 108 TU / ml, si RNA-4 2.31 × 108TU / ml, the virus titer of negative control group carrier packaging: 1.79 × 109 TU / ml. The efficiency of the four kinds of recombinant lentiviral vector to infect MRC-5 cells were about 93%. Compared with normal control group and negative control group, the expression of TGFβRⅠmRNA in MRC-5 cells was significantly decreased (P = 0.000) after infected with 4 recombinant lentiviral vector (si RNA-1 ~ 4) si RNA-2 decline the most obvious, the best efficiency of silencing. The result of Western blotting was consistent with that of q RT-PCR. The expression of TGFβRI in MRC-5 cells was significantly decreased by all four recombinant lentiviral vectors (P = 0.000), and the interference efficiency of TGFβRⅠ-si RNA-2 was the highest. Conclusion: The best RNAi recombinant lentiviral vector, TGFβR-si RNA-2, which can effectively silence TGFβRⅠ gene, was screened and constructed, which laid the foundation for further study.