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目的利用bcl-x微基因(minigene)构建重要顺式作用元件B2G、CRCE2的突变微基因,建立bcl-x微基因报告法并应用于bcl-x选择性剪接机制的研究。方法以构建成功的pcDNA3.1(-)-bcl-x微基因为模板,利用反向PCR的方法,按碱基互补配对原则设计能让顺式元件B2G,CRCE2定点突变的引物,进行PCR反应。PCR反应结束后,用DpnⅠ对模板质粒DNA进行消化,然后使PCR产物发生自身环化反应。将环化产物转化入感受态DH5α,通过筛选并最后利用测序来鉴定所构建突变微基因成功与否。结果测序结果鉴定,所克隆的突变微基因序列碱基无一错误突变。并且在HL-60细胞中,RT-PCR实验表明bcl-x顺式作用元件B2G,CRCE2对bcl-xL/bcl-xS有一定影响,pcDNA3.1(-)-bcl-x-B2G(M)转染后RT-PCR电泳显示bcl-xS几乎消失,pcDNA3.1(-)-bcl-x-CRCE2(M)转染后RT-PCR电泳显示bcl-xL/bcl-xS为1.6。结论成功构建及应用人类凋亡相关基因bcl-x的突变微基因,顺式作用元件B2G缺失使bcl-xS几乎消失,CRCE2突变使bcl-xL/bcl-xS上升。
OBJECTIVE: To construct the bcl-x microgene reporter gene by using the bcl-x minigene to construct the mutant minigene of important cis-acting elements B2G and CRCE2 and to apply it to the study of bcl-x alternative splicing mechanism. Methods Using the constructed pcDNA3.1 (-) - bcl-x microgene as a template, reverse PCR was used to design primers that allow for the site-directed mutagenesis of the B2G and CRCE2 cis elements according to the principle of base pairing. . After completion of the PCR reaction, the template plasmid DNA is digested with DpnI, and then the PCR product undergoes a self-cyclization reaction. The cyclized product was transformed into competent DH5α, and the success or failure of the constructed mutant micro-gene was identified through screening and finally sequencing. Results The sequencing results identified that there was no missense mutation in the cloned gene sequences. And in HL-60 cells, RT-PCR experiments showed that the bcl-x cis-acting elements B2G and CRCE2 have certain influence on bcl-xL / bcl- xS, pcDNA3.1 (-) - bcl-x-B2G After transfection, the bcl-xS almost disappeared by RT-PCR and the bcl-xL / bcl-xS was 1.6 by RT-PCR after pcDNA3.1 (-) - bcl-x-CRCE2 (M) transfection. Conclusion The mutation of bcl-x gene in human apoptosis gene was successfully constructed and applied. The deletion of cis-acting element B2G almost abolished bcl-xS, while the mutation of bcl-xL / bcl-xS increased with CRCE2 mutation.