β-thalassemia for translating human β-globin gene and RNA interference of α-globin gene expression by lentivirus vectors
نویسندگان
چکیده
Objective: According to the characteristics of α/β-globin gene imbalance caused by α-globin gene accumulation in β-thalassemia, we designed two lentiviral vectors, that is, while we used the lentiviral to mediate full length human β-globin gene, we introduced the interference fragment of lentiviral α-globin gene to adjust the α/β chain ratio by increasing the expression of β-globin and decreasing the expression of α-globin. Methods: We gained lentiviral vectors LV-β-globin by extracting DNA of β-globin expresser. According to the design principles of RNA interference sequence, the optimal dynamic parameters of RNA interference target sequence are selected based on the design experience and software evaluation. After lentivirals were stably transfected into K562 cells, the total RNA was extracted, and the expression of m RNA of α-(HbA2) and β-(HBB) globin gene in K562 cells was detected by PCR real-time. Western blotting was used to detect the expression of alhpa and β-globin. Results: β-globin gene was amplified to specific band of 227 bp by RT-PCR, and the expression of RT-PCR α-globin gene was amplificated to specific band of 85 bp as well. 2-ΔΔCt value of β-globin gene mRNA was 4.080 ± 0.078 and 2-ΔΔCt value of α-globin gene mRNA was 0.274 ± 0.023. Results of western blotting test indicated that in the stably transfected K562 cells, the synthesis of human β-globin was significantly higher than that of the blank group and negative control group and the synthesis of α-globin was significantly lower than that of the blank group and negative control group. Western blotting showed that the grey level of β-globin was 1.063 ± 0.082, and the grey level of α-globin was 0.327 ± 0.042, with statistical significance. Conclusions: This study successfully transfected β-globin gene and α-globin gene siRNA into K562 cell. After lentiviral transfection, the levels of β-globin gene RNA and protein were significantly higher than the control group.
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