摘要
目的克隆人血管紧张素转换酶2基因(ACE2),并构建其真核表达载体。方法用Trizol试剂从人肺组织提取总RNA,然后经逆转录得到人ACE2的cDNA。半巢式PCR方法扩增人ACE2基因后,先进行T-A连接,转化感受态大肠杆菌DH5α,接种含IPTG和x-Gal平板筛选重组子,并进一步亚克隆至真核表达质粒载体pcDNA3.1(+)。经PCR扩增、酶切和序列测定方法鉴定重组质粒。结果获得了人ACE2的编码基因,并成功构建了其真核表达载体。结论人血管紧张素转换酶2是SARS冠状病毒的功能受体,与SARS冠状病毒Spike蛋白上的受体结合域相结合,在SARS冠状病毒感染和传播中起重要作用,本工作为研究SARS冠状病毒感染及跨种属传播机制奠定了基础。
Objective To clone the human angiotensin-converdng enzyme 2 （ACE2）and construct its eukaryotic expression plasmid. Methods Total RNA was extracted from the human lung tissue using Trizol reagent, and the human ACE2 gene was amplified by Semi-Nest RT-PCR, and cloned into the T-vector. The ACE2 cDNA was then subcloned into eukaryotic expression vector pcDNA3.1 （＋）. Positive recombinants were identified with restriction enzyme digestion, PER amplification and DNA sequencing. Results Human ACE2 cDNA was successfully amplified and its eukaryotic expression plasmid was constructed. Conclusion Human ACE2 is a functional receptor of SARS Coronavirus interacting with receptor binding domain of the Spike protein and plays an important role in the pathogenesis of the disease. This work provides the basis of further study on interspecies transmission molecular mechanism.
出处
《热带医学杂志》
CAS
2006年第7期776-778,共3页
Journal of Tropical Medicine
基金
广州市科技局应用基础项目(No.2004J1-C0211)
广州市教委科研项目(No.1040
1047)
广州医学院科研项目(No.03-K-25)
