BCR/ABL重组质粒实时荧光定量PCR标准品的制备

Preparation for the Standard Plasmids of BCR/ABL Fused Genes with Real-Time PCR

目的:用T-A克隆法构建含BCR/ABL融合基因的重组质粒,并用实时定量PCR(RQ-PCR)方法制备标准品。方法:通过培养细胞,提取总RNA并逆转录为cDNA后做PCR,电泳胶回收纯化,T-A克隆与pUCm-T载体连接,转染DH5a菌,蓝白斑筛选阳性菌落后,大量提取质粒,再进行RQ-PCR,最后制得BCR/ABL的重组质粒标准品。结果:蓝白斑筛选实验、PCR扩增均证实BCR/ABL融合基因重组到pUCm-T载体上,经RQ-PCR定量后得到BCR/ABL重组质粒标准品的标准曲线。结论:该方法能大量制备质粒标准品,并且可被推广应用。

Objective To construct recombinant plasmid containing BCR/ABL with T-A clone,and to preparate standard plasmids with real-time quantitative polymerase chain reaction（RQ-PCR）.Methods BCR/ABL fused gene was obtained with RT-PCR from mRNA retreived from chronic leukemia K562 cell-line culture.The product was conjugated into pUCm-T vector with FA cloning and transfected into DH5a E.coli.The plasmid from white positive colonies was furthur amplified with RQ-PCR and standard preparation was obtained.Results The blue/white blot screening experiment and PCR amplification confirmed that the BCR/ABL fused genes was recombined with pUCm-T vector,and the standard curve of recombinant standard plasmids was obtained.Conclusion This method could yield guantities of standard plasmids and might deserve popularization.