酪氨酸羟化酶重组质粒标准品构建

目的:用T-A克隆法构建含酪氨酸羟化酶(TH)基因的重组质粒,并用实时定量PCR(RQPCR)方法制备标准品。方法:提取小鼠肾上腺髓质组织总RNA,逆转录为cDNA后进行RT-PCR,回收纯化电泳胶,T-A克隆与pGEM-T载体连接,转化Top-10感受态细菌,蓝白斑筛选出阳性菌落后,大量提取质粒,再进行RQPCR反应,最后制得含TH的重组质粒标准品。结果:肾上腺髓质TH的表达,PCR扩增,菌液PCR,测序鉴定均证实TH重组到pGEM-T载体上,经RQPCR定量后得到TH重组质粒标准品的标准曲线。结论:该方法能大量制备TH质粒标准品,并且可推广应用。

Construction of Tyrosine Hydroxylase Recombinant plasmids

Objective:To construct recombinant plasmid containing Tyrosine hydroxylase(TH) with T-A clone,and to prepare standard plasmids with real-time quantitative polymerase chain reaction(RQPCR).Methods: TH gene was obtained with RT-PCR from mRNA retreived from adrenal medulla.The product was conjugated into pGEM-T vector with T-A cloning and transformmed into Top-10 E.coli.The plasmid from white positive colonies was furthur amplified with RQPCR and standard preparation was obtained.Re-sults:The bacterial PCR and PCR amplification confirmed that the TH genes were recombined with pGEM-T vector,and the standard curve of recombinant standard plasmids was obtained.Conclusion:This method could yield guantities of standard plasmids of TH and might deserve popularization.