Expression Of SARS-Cov Spike Protein Fragment Of S144-643 And Its Critical Biological Trait Investigation

Severe acute respiratory syndrome(SARS) is called atypical pneumonia in China, characterized by progressive respiratory failure and death in about 10% of cases. It was recognized for the first time in Guangdong Province of China in November 2002, then spread to 32 countries and regions rapidly. The causative agent of SARS has been identified as a novel coronavirus(SARS-associated coronavirus, SARS-CoV) which does not belong to any of the previously identified three groups. Based on the fact that few drugs are effective to treat SARS, several strategies are being investigated to prevent and treat SARS including blocking the binding of virus and its receptor by simulating receptor molecules or viral receptor binding proteins, inhibiting the fusion of virus and cell membranes by small peptides, developing subunit vaccines using specific neutralizing epitopes. The S1 domain of SARS-CoV spike (S) protein, containing the functional receptor binding domain of 193 amino acids (residues 318-510) and several neutralizing epitopes, binds its functional receptor of angiotensin converting enzyme 2(ACE2). In this study, the cloning and expression of the receptor binding domain of SARS S1 protein were performed to provide the tool to identify the functional domain of SARS-CoV receptor ACE2. Moreover, screening the specific binding peptide from random Phage Display Peptide Library using the expressed S1 protein as the target provides evidence for development of antivirus agents and investigation of the interaction between S1 protein and its antibodies.In this study,the gene fragment of S1 protein S144-643 (nt430-nt1929) was subcloned into pET-28b and expressed in E.coli. The protein with His-tag,, about 50KDa, existed mainly in bacterial inclusion bodies. Western blot assay demonstrated the recombinant protein S144-643 was specifically recognized by the sera from convalescent SARS patients. Furthermore, the S144-643 protein was able to induce high titer of specific antibodies in immunized New Zealand rabbits. The fact above revealed that the expressed S144-643 protein in E. coli was immunologically active. Furthermore, it was confirmed with Western blot assay that S144-643 was able to bind to the viral receptor of ACE2 protein. Combined with the data of ACE2 obtained by Yang Guoliang,, it was concluded that the N terminal 335 amino acids of ACE2 contains SARS-CoV S1 binding domain.The S144-643 was purified with His.Bind Purification Kit, and was used as the target to screen the specific binding peptide from Ph.D.-12 Phage Display Peptide Library. After 3 rounds of screening, 10 positive phage clones were identified with the methods ofphage ELISA and Dot-blot. The sequencing result showed there is distinct conserved sequence among 10 positive phage clones. Using the pseudoviruses system, it was primarily demonstrated that two positive phage clones were able to neutralize viral infectivity in cultured cells.