Expression Of The Receptor Binding Domain Of Bat Sars Like Coronavirus Spike Protein In Bac-to-bac Baculovirus Expression System

AIMS:The spike protein (S protein) on the surface of SARS coronavirus can induce virus entering into the host cell by means of binding to the Angiotensin-converting enzyme 2 (ACE2), the virus receptor on the cell. The receptor binding domain (RBD) of S protein is the smallest functional fragment that binds to ACE2 stably, which plays a key role in the inter-host transmission of the virus.In studies of source-tracing of the SARS coronavirus, researchers found the bat SARS-like coronavirus (Bat SARS-LcoV) from bats. However, it can not be amplified and isolated in the culture cells eventually, which will limit the studies of the cross-species transmission mechanisms because no live virus can be used for infecting cells and animal models directly. In this study, Spike protein RBD fragment of the bat SARS-like coronavirus (Bat SARS-LcoV) was recombinanted and expressed in the baculovirus expression system. And the kinetics of gene expression and purification conditions were studied to provide the experimental basis for the further researches of the possible spread routes among the animals and the molecular mechanisms of the cross-species transmission of the Bat SARS-Like-CoV. It aslo can provide fundamental material for further study the antigenic and immunogenic of the Spike protein from Bat SL-CoV. Methods:1. Constructing the donor vector pFAST-Bat-RBD: designing primers according to the 964-1542 fragment of the Bat SARS-LcoV spike gene (published in the Genbank) . Supplementing FLAG-Tag gene in the reverse primer. The gene of interest was amplified by PCR and was linked to pMD18-T vector. And then the gene was subcloned into the vector pFAST-HTB to construct the recombinant donor vector : pFAST-Bat-RBD.2. Generating recombinant baculovirus and determining the virus titer: tranforming pFAST-BAT-RBD into E.coli DH10Bac competent cell, and the transpositional recombination would take place between BAT-RBD and the baculovirus genome (Bacmid) in E.coli DH10Bac cells. Transfecting the sf9 insect cells with the recombiant Bacmid-BAT-RBD to generate the recombinant baculovirus. And then determining the virus titer by 50% Tissue culture infective dose (TCID50).4,The Expression and assay for the recombinant protein BAT-S-RBD: infect the insect cells with the high titer virus stock at suitable multiplicity of infection (MOI). Harvest cells and medium at 72 hours after infection and perform SDS-PAGE or western blot analysis to assay expression of BAT-S-RBD. Purify the recombinant protein by using nickel chelating resin.Result:1. The results of PCR, double digestion and sequencing confirmed that the cloned gene had been inserted into plasmid correctly, its sequence was matched with the 964-1542 fragment of the bat SARS-like virus S protein gene, which demonstrated that the plasmid pFAST-Bat-RBD was successfully constructed.2. The PCR results indicated that the target gene had been correctly recombined into the baculovirus genome. The cells transfected with viral genome caused typical cell pathological changes, and the diluted culturing supernatant could cause the same changes to the normal cells, which showed that the recombinant virus produced successfully. The titer of P1 virus stock was 8.6×10~6 PFU/ml, P2 virus stock was 5.3×10~7 PFU/ml and P3 virus stock was 2.1×10~8 PFU/ml,which converted from 50% Tissue culture infective dose (TCID50).3. The results of SDS-PAGE and Western Blot showed that the recombinant protein BAT-S-RBD only expressed in cells but not secreted into the medium. The most appropriate MOI of the recombinant protein expression was 3. Peak expression of the protein occurred between 56 hours and 64 hours after infection by P3 virus. The recombinant protein BAT-S-RBD could be purified by nickel-chelating resin.Conclusion:This study have successfully constructed the recombinant baculovirus containing the target gene which can be effectively expressed in insect cell lines. The expression and purification conditions of the recombinant protein have been initialy optimized. The study provides the experimental basis for the further researches of the possible spread routes among the animals and the molecular mechanisms of the cross-species transmission of the Bat SARS-Like-CoV. It aslo can provide fundamental material for further study the antigenic and immunogenic of the Spike protein from Bat SL-CoV.