巢式PCR法构建人细胞色素P4501A1基因cDNA全长T载体

Cloning the whole cDNA of human cytochrome 1A1 gene by nest- PCR

目的构建含限制性内切酶位点KpnI,XhoI的人细胞色素氧化酶(CYP1A1)基因cDNA全长的pGEM -T载体。方法从培养的MCF-7细胞中抽提总RNA并根据人细胞色素P4501A1(CYP1A1,GeneBank NM- 000499)开放阅读框设计两对引物,采用巢式-PCR方法扩增CYP1A1 mRNA全长,凝胶分离并回收扩增的DNA片断,与T载体连接后转化大肠埃希菌DH5α,挑选3个阳性克隆,扩增培养后抽提重组质粒并进行PCR及酶切鉴定, 再行测序分析。结果重组质粒PCR扩增得到1568 bp的片段,KpnI,XhoI限制性内切酶消化质粒证实目的片断成功插入至载体,测序分析也进一步证明了目的片段与GeneBank中CYP1A1 mRNA的序列同源性为99.9%。结论该实验成功地构建了含CYP1A1基因cDNA全长区域的T载体克隆,巢式-PCR是一种简便、特异、灵敏的方法, 可用于基因的检测和载体的构建。

Objective To clone and construct the pGEM - T vector containing the whole open read frame（ORF） of human cytochreme 1A1 gene with two recognition sites for the enzymes KpnI and XhoI. Methods Using total RNA extracted from human MCF - 7 and primers were designed according to the open reading frame regions of Home sapiens cytochrome P450 1A1 （cytochrome 1A1, GeneBank NM-000499）. The full- length cDNA of CYP1A1 mRNA was cloned by nest- PCR. The amplified DNA fragments were separated and recovered from agar gel, subsequently hgated into pGEM- T vector, and transformed into E. coil strain DH5α. Three positive clones were screened out and recombinant plasmids were isolated from such colonies. The recombinants were identified by PCR, restriction analysis and sequencing. Results PCR assay showed that a DNA fragment of 1 568 bp could be amplified. Restriction endonucleases （KpnI and XhoI） digestion confirmed the target fragment has been inserted into pGEM- T successfully. The sequencing results verified that the target fragment had 99.9% homelogy with the CYP1A1 mRNA sequence in Genebank. Conclusion The T- vector clone with the whole cDNA of cytechrome 1A1 gene has been successfully constructed. Nest - PCR is simple, effective and special for detection of gene and construction a variety of useful T -vector.