Construction And Panning Of Immune Phage Antibody Library Against SARS-CoV

Severe acute respiratory syndrome (SARS) has emerged as a frequently fatal respiratory-tract infection caused by the newly identified SARS coronavirus .After the worldwide SARS epidemic in 2002-2003, sporadic cases continued to arise in southern China, possibly because of human contact with the animal reservoir. Two cases of laboratory acquired SARS coronavirus infections in China last year spread into the community and triggered extensive efforts in tracing and isolating contacts of patients to prevent a new epidemic. These indicate that SARS epidemics may recur at any time in the future. Means to control SARS coronavirus infection through active or passive immunisation are, therefore, urgently needed. In our investigation, we used phage display technique to isolate human antibody against RBD of SARS-CoV spike. Acquirement of antibodies lays the foundation of effective prophylaxis , diagnosis and antiviral therapy, moreover, the highly potential neutralizing antibody implies the possibility of developing subunit vaccine to prevent SARS.There are four parts of our study as below:Part 1 Expression of RBD of SARS-CoV spike protein in E. coli.The 193-amino acid RBD (residues 318-510) was expressed in E.coli. inorder to screen neutralizing antibody efficiently by reducing interference of other non-neutralizing sites. SARS-CoV s rbd gene was amplified by RT— PCR , cut by Nde I and Xho I and inserted to prokaryotic expression vector PET30b(+). The expressed product with His-Tag induced by IPTG was purified by Ni-NTA Magnetic Agarose Beads and identified by SDS-PAGE, ELISA and Western blot. The results showed that RBD of SARS-CoV spike protein was expressed in E.coli. JM109(DE3);purity of the protein was >90% and it could bind to convalescent serum of SARS patients. The expression of RBD of SARS-CoV spike protein lays the foundation of acquirement of neutralizing human antibodies.Part 2 Construction of human Fab immune phage antibody libraryLymphocytes were isolated from blood of people who ever were infected with SARS-CoV. Total RNA was extracted from lymphocytes. Genes of L chain and Fd of antibodies were amplified by RT-PCR and inserted to phagemid vector Pcomb3x. Ten clones were randomly selected to identify recombinant ratio by digesting with restriction endonucleases. The results showed that the phage antibody library was successfully constructed ;capacity of library was 6.2 × 10~7 and recombinant ratio was 75%.Part 3 Isolation of human Fab antibody against RBD of SARS-CoV spike proteinThe antibody library was rescued by helper phage M13K07 firstly, then was used to screen RBD of SARS-CoV spike protein coated on ELISA plate. The screening repeated three cycles with 2-fold dilution coated antigen from 20μg/ml to 5μg/ml. After the third round, 220 clones were rescued to perform Phage—ELISA. Nine positive clones were then selected to express in E.coli. The expressed products were identified by Western blot. The results...