SARS-CoV S1 Gene Expression In Pichia Pastoris AND Study On Inhibitory Peptides

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 explain the viral pathogenesis and antivirus drug. Moreover, screening the specific binding peptide from random Phage Display Peptide Library using the expressed S1 protein, as inhibitive peptides.Modified S1gene containting receptor binding region was amplified by several primers though Over-Lap PCR. the resultant gene sequence was changed at AT repeated regions (777-1683,1041-1050, 1236-1248, 1317-1335, 1590-1605) without the amino acid replacement, and cloned into yeast expression vector pPIC9K. Resultant plasmid pPIC9K- S1 was transformed to P. pastoris GS115, the supernatant by SDS-PAGE and Western Blot suggested S1 gene expression in P. pastoris GS115. The expression level of this recombinant S1 protein was about 69mg/L using UV check, suggesting P. pastoris GS115 is a good system for S1gene. Purified recombinant S1 protein was used subsequently as an ELISA antigen for detection of eight 6 serum samples, results suggest that the recombinant S1 protein may be useful as a diagnostic reagent. In Western blot, S1 protein can specially bind ACE2 , suggesting S1 protein expressed in P. pastoris GS115 is a good material to study primary receptor ACE2 in SARS-CoV infection.The S259-561 was purified with saturated ammonia sulfate, 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, 8 positive phage clones were identified with the methods of...