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目的建立中华菊头蝠体内扩增血管紧张素转换酶2(ACE2)基因真核表达载体,研究SARS冠状病毒感染的跨种属传播机制。方法利用基因的种属保守性分别设计用于扩增5’未端和3’端cDNA的引物,从中华菊头蝠小肠组织中提取总RNA,在通过cDNA末端快速扩增(RACE)技术获得cDNA 5’端和3’端序列基础上,进一步PCR扩增ACE2开放阅读框并克隆到pcDNA3.1。结果成功获得了中华菊头蝠的ACE2基因全长序列,并构建了其真核表达载体pcDNA3.1/R-ACE2。结论利用RACE技术扩增并构建了中华菊头蝙蝠的ACE2的真核表达载体,为研究SARS冠状病毒感染与免疫及跨种属传播机制奠定了基础。
Objective To establish an eukaryotic expression vector of angiotensin-converting enzyme 2 (ACE2) gene in Chinese chrysanthemum, and to study the mechanism of cross-species transmission of SARS-CoV infection. Methods The primers used to amplify the 5 ’and 3’ cDNAs were designed according to the conservation of the gene species. Total RNA was extracted from the small intestine tissues of the head bats, and was obtained by rapid amplification of cDNA ends (RACE) Based on the 5 ’end and the 3’ end of the cDNA sequence, the ACE2 open reading frame was further amplified by PCR and cloned into pcDNA3.1. Results The full-length sequence of ACE2 gene was successfully obtained and its eukaryotic expression vector pcDNA3.1 / R-ACE2 was constructed. Conclusion The eukaryotic expression vector of ACE2 of Chrysolophus pictus was amplified and constructed by RACE technique, which laid the foundation for the study of SARS-CoV infection and immune and trans-species transmission mechanism.