The Establishment Of SARS Susceptible Transgenic Mice Which Express The SARS Coronavirus Functional Receptor Human ACE2

The host of SARS coronavirus is restricted to humanbeing and a few kinds of primates, the lack of appropriate animal model impeded a more thorough understanding of SARS and SARS infection associated disease. According to recent research of SARS coronavirus, human ACE2(hACE2)was ascertained as the functional receptor of severe acute respiratory syndrome associated coronavirus (SARS-Cov). When hACE2 was transfected and expressed in various cell lines, SARS-Cov could infect and replicate in these cells even though the parent cell never been susceptible to SARS-Cov.Transgenic mice expressing human virus receptor have been used as animal model for the evaluation of antiviral agents and vaccines. In our study, we try to generate transgenic mice model that express SARS-Cov functional receptor hACE2 to substitute the primates model such as monkey.First, the cDNA coding for hACE2 was amplified by PCR and then cloned into a eukaryotic expression vector PCAGGS, under the control of the CAG promoter, a composite promoter consisting of the cytomegalovirus immediate-early enhancer, mice NIH3T3 cells were transfected and subjected to G418 selection, the positive clones were screened and the integration of foreign DNA fragments was identified by PCR and Southern blot analysis. The high-level expressed hACE2 in NIH3T3 cells was verify by western blot, FACS, and immunofluorescence method, the ability of binding with S protein of SARS-Cov for the expressed hACE2 was also identified.For the establishment of transgenic mice, two expression vector PCAGGS-hACE2 and pcDNA3.1+-hACE2 were constructed. The purified 5.5-kb DNA fragment generated by an SalI and DrdI double digestion of PCAGGS-hACE2 and the purified 6.5-kb DNA fragment generated by an Bst1107I and pvuI double digestion of pCDNA3.1+-hACE2 were used for microinjection.12 PCAGGS-hACE2 transgenic founders and 10 pcDNA3.1+-hACE2 transgenic founders were get and identified by PCR and Southern blot analysis, the transgenic positive rate were 12.5% and 9.1% individually. These founders were backcrossed with wild-type KM mice and set lineages. The expression of exogenous hACE2 in these transgenic mice were tested, the total RNA of mice lungs were extracted and then be subjected to RT-PCR analysis, positive results were get for the PCAGGS-hACE2 transgenic mice while the negative for the pCDNA3.1+-hACE2 transgenic mice. These results indicated that the PCAGGS expression vector is more appropriate for the expression of foreign gene in mice. Then the total protein of lung tissue for PCAGGS transgenic mice was extracted for the western blot analysis, results showed that hACE2 had been strongly expressed in the lung tissue of transgenic mice. The lungs of transgenic mice were sliced for Immunofluorescence staining, and results showed the hACE2 expressed in the mice lung can bind the S protein of SARS-Cov while the negative results were get for the wild-type KM mice.Until now We get the transgenic mice that express severe acute respiratory syndrome associated coronavirus (SARS-Cov) functional receptor human ACE2(hACE2). It will provide an useful animal model for the research of SARS-Cov infection.