SARS-CoV Receptor ACE2 From African Green Monkey Kidney Cells, CDNA Cloning, Expression And Identification Of Receptor Function Domain

The worldwide outbreak of atypical pneumonia during later 2002 and early 2003 made a tremendous impact on human life, and was paid the great attention by the scientists in the world. This emerging disease is a severe infectious respiratory disease, designated as severe acute respiratory syndrome (SARS) by WHO. A distinct novel coronavirus, SARS-CoV, had been identified as the aetiological agent of SARS. Although CD209L was recently determined to be another receptor, angiotensin-converting enzyme (ACE2), a carboxypeptidase and an essential regulator of heart function, was the major functional receptor of SARS-CoV. This thesis was aimed to clone and identify ACE2, identify the functional domain of ACE2, and provide evidence to further study the interaction between SARS-CoV and host cells, the interspecies infection of SARS-CoV and development of antiviral agents.1. Expression of ACE2 in Pichia pastorisTwo sets of specific primers were designed to amplify ACE2A and ACE2B fragments from the whole mRNAs of Vero-E6 cells with one-step RT-PCR. The amplified products were cloned into Pichia pastoris expression vector pPIC9K to construct recombinant plasmids p9K-ACE2A, and p9K-ACE2B. The constructs were linearized and further transformed into LiCl treated competent Pichia pastoris strain GS115. After induced by methanol for 5 days, the recombinant strains secreted the soluble proteins in the supernatant of culture media. The molecular mass was determined to be 41KD and 52KD correspondingly with SDS-PAGE. Dot-blot assay showed that both proteins displayed good immune responses to specific antibodies. This work laid a good foundation for the study of interaction between SARS-CoV and the cell receptor.2. Expression of ACE2 in E. coli and identification of its functional domainTwo fragment of ACE2 (ACE2A and ACE2B) were inserted into pGEX-KG, and resulted in the constructs pGEX-ACE2A and pGEX-ACE2B respectively. Both of the two gene fragments were fused to GST gene in the constructs. Recombinant plasmids were transformed into ￡. coli BL21(DE3), and the protein expression was induced by IPTG (Isopropyl-b-D-Thiogalactoside). High level expression of the recombinant proteins were confirmed by SDS-PAGE. The molecular masses of the two fusion proteins were respectively 65KD and 77KD. Western blot analysis demonstrated that the recombinant proteins had immunological activity. The protein ACE2A (65KD) but not ACE2B (77KD) could bind to S1 protein of SARS-CoV. This result showed that the receptor activity of ACE2 was not related to the enzyme activity site, and the receptor functional domain was within N terminal 335 amino acids. This was so far the shortest ACE2 fragment expressed which had SARS-CoV receptor function.3. Investigation of functional domain of SARS-CoV spike protein by phage displayPhage display was used in this thesis to screen for short peptides that bind to ACE2A. Purified fusion protein GST-ACE2A 100|xg/ml was coated on wells of ELISA plates at 4°C overnight, then blocked with 0.1M NaHC03 (pH 8.6) containing 5mg/ml BSA for lh. For the first round of panning, lOul 12-mer random phage library was added to each well. GST bound phage was excluded by panning the phage library with GST protein. Then the ACE2 specific-binding phage was concentrated after a three-bound panning with GST-ACE2A.Twenty plaques were randomly picked up, and amplified. The phage DNA was sequenced with the recommended primers. A total of seven peptide sequences were identified. Alignment analysis of the peptide sequences with spike protein of SARS-CoV with BLAST, two peptides of them share some identical sequences in the binding domain (S318-510) of the spike protein to ACE2. Probably, it is of significance to develop antiviral drugs and vaccines.