Identifying Inhibitors Of The SARS Coronavirus Membrane Fusion,Prokaryotic Expression Of S2 Subunit And Production Of Polyclonal Antibodies

Objective: In these studies we use docking techniques to study if chemical compounds which inhibit HIV membrane fusion have the same ability against fusion of SARS-CoV with target cells. We have cloned, expressed the S2 subunit of spike S glycoprotein, and at the same time, the polyclonal antibodies which can specifically recognize S2 subunit were produced. Methods: We searched and downloaded the chemical compounds against HIV membrane fusion in PubChem database. Docking calculations were carried out by using DOCK version 5.1.1 to predict the binding affinities of SARS-CoV central HRl coiled coil structure and HIV fusion inhibitors. We amplified S2 fragment by using PCR technique and sub-cloned it into pGEX-5X-3 expression vector. Then, the pGEX-5X-3 -S2 plasmid was transformed into E. coli strain BL21. Protein expression was induced by addition of 1.5mmol/L IPTG. After SDS-PAGE analysis of the expression protein, we cut it to immunize rabbit. Finally Agarose Double immunodiffusion was taken to detect the rabbit serum. Results: We found the ADS-J1, ADS-J2, XTT Formazan have binding affinities for SARS fusion core, because they obtained high score in docking calculations. The plasmid pGEX-5X-3-S2 wasreconstructed successfully and a new added 84.2KD fusion protein was detected by 10% SDS-PAGE. Agarose Double Immunodiffusion analysis of rabbit serum showed the polyclonal antibodies had been developed. Conclusion: Our study indicates that existing HIV membrane fusion inhibitors have the same activities to SARS-CoV. We have successfully expressed the S2 subunit in Escherichia coli BL2, and polyclonal antibodies which could recognized S2 subunit specifically have been developed.