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β-珠蛋白基因第2个内含子(IVS-2)中第654位核苷酸(nt654)C→T剪接缺陷型突变是中国人最常见的β-地中海贫血(简称β-地贫)突变类型之一.我们先前的研究表明,该突变在IVS-2第654位(nt654)上出现的单碱基取代产生了一个新的 GT二核苷酸,它联同旁侧序列构成了一个5＇异常剪接位点,同时还激活了IVS-2第579位(nt579)处3＇隐匿剪接位点,从而将 IVS-2nt580至 nt652之间一段73bp内含子序列作为额外的外显子插入外显子2和外显子3之间,产生了异常的β-珠蛋白mRNA,导致β~+-地贫.为了探讨反义核酸治疗β-地贫IVS-2-654 C→T突变的可能性,我们应用体外转录和体外剪接系统,以体外转录产生的特异反义RNA封闭IVS-2-654 C→T突变基因中3＇隐匿剪接位点和5＇异常剪接位点,使之失去活性,从而减少异常加工的mRNA并且部分恢复了正常剪接途径.
The 654th nucleotide (nt654) in the second intron of the beta-globin gene (IVS-2) C → T splice-deficient mutations are the most common β-thalassemia in Chinese people (beta-thalassex) Our previous study showed that the single base substitution of this mutation at position 654 (nt654) of the IVS-2 gene resulted in a new GT dinucleotide, which together with the flanking sequences constitutes a 5 ’anomalous splice sites and also activate the 3’ conceal splice site at 579 (nt 579) of IVS-2 to insert a 73 bp intron sequence from IVS-2 nt580 to nt652 as an additional exon Aberrant β-globin mRNA was produced between exon 2 and exon 3, resulting in β ~ + -thalassemia. To explore the role of antisense nucleic acids in the treatment of β-thalassemia subtypes IVS-2-654 C → T mutations Possibility, we use in vitro transcription and in vitro splicing system, in vitro transcription of specific antisense RNA blocking IVS-2-654 C → T mutations in the 3 ’occluder site and the 3’ site of abnormal splice, so that it lost Activity, thereby reducing abnormally processed mRNA and partially restoring the normal splicing pathway.