| The outbreak of SARS in 2003, has done much impair on the human health and the social economy. Many scientific researches of the world have been studying the pathway of SARS-CoV invading the host cells , in order to find the vaccine which can prevent the human from the infection of SARS-CoV or the effective therapy drugs for the patients. Based on the researching results of our department that Cyclophilin A is on the surface of SARS-CoV particles and the antagonist of CD147 can inhibit the invading of SARS-CoV, and according to the results that HIV-1 entry depends on an interaction between virus-associated CyPA which binds to the CA domain of Gag precursor polyprotein and CD147 on a target cell, we want to confirm the hypothesis that CyPA which binds to the nucleocapsid (N) protein of SARS-CoV and CD147 enhances the early step of cellular SARS-CoV infection.Three steps are included in the experiments process: 1. Cloning the correct cDNA of CyPA protein and the structural protein of SARS-CoV. 2.Constructing the prokaryotic expressing vectors of N and CyPA protein and purifying the target proteins. 3. Researching the interaction between N and CyPA protein . We got the cDNA of N protein from the particles of SARS-CoV and CyPA protein from the human lymphocyte by RT-PCR, and cloned them into the prokaryotic expressing vector of pET-25b(+). We purified the bioactivity proteins which purity is >90% according to their characters. And we confirmed their relationship by the experiments of West-blotting, Coimmunoprecipation and BIAcore. The result showed that we got the correct cDNA of N and CyPA protein, and got the purified bioactivity proteins , at last we confirmed that the N protein can bind to the CyPA protein . Conclusion: the hypothesis that CyPA which binds to the nucleocapsid (N) protein of SARS-CoV and CD147 enhances the early step of cellular SARS-CoV infection was confirmed by the techniques of molecular biology and biophysics, and the KD of N and CyPA is 5. 07 X 10-6 M.
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