AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion ...AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.展开更多
Two-dimensional(2D)metal oxides(2DMOs),such as MoO_(2),have made impressive strides in recent years,and their applicability in a number of fields such as electronic devices,optoelectronic devices and lasers has been d...Two-dimensional(2D)metal oxides(2DMOs),such as MoO_(2),have made impressive strides in recent years,and their applicability in a number of fields such as electronic devices,optoelectronic devices and lasers has been demonstrated.However,2DMOs present challenges in their synthesis using conventional methods due to their non-van der Waals nature.We report that KCl acts as a flux to prepare large-area 2DMOs with sub-millimeter scale.We systematically investigate the effects of temperature,homogeneous time and cooling rate on the products in the flux method,demonstrating that in this reaction a saturated homogenous solution is obtained upon the melting of the salt and precursor.Afterward,the cooling rate was adjusted to regulate the thickness of the target crystals,leading to the precipitation of 2D non-layered material from the supersaturated solution;by applying this method,the highly crystalline non-layered 2D MoO_(2)flakes with so far the largest lateral size of up to sub-millimeter scale(~464μm)were yielded.Electrical studies have revealed that the 2D MoO_(2)features metallic properties,with an excellent sheet resistance as low as 99Ω·square^(-1 )at room temperature,and exhibits a property of charge density wave in the measurement of resistivity as a function of temperature.展开更多
文摘AIM： To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus （SARS-Cov）, and to analyze its receptor binding ability. METHODS： Three fusion tags （glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP）, which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli （BL21（DE3） and Rosetta-gamiB （DE3） strains）. The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS： RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 （DE3） and Rosetta-gamiB （DE3） under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21（DE3）, and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST.RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD＇s binding ability to ACE2 （angiotensin-converting enzyme 2） positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST.RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION： In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in EcoliBL21 （DE3）; data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.
基金supported by the National Key Research and Development Program of China(Nos.2023YFB3608703 and 2023YFB3608700)Fujian Science&Technology Innovation Laboratory for Optoelectronic Information of China(Nos.2021ZZ122 and 2020ZZ110)Fujian provincial projects(Nos.2021HZ0114 and 2021J01583).
文摘Two-dimensional(2D)metal oxides(2DMOs),such as MoO_(2),have made impressive strides in recent years,and their applicability in a number of fields such as electronic devices,optoelectronic devices and lasers has been demonstrated.However,2DMOs present challenges in their synthesis using conventional methods due to their non-van der Waals nature.We report that KCl acts as a flux to prepare large-area 2DMOs with sub-millimeter scale.We systematically investigate the effects of temperature,homogeneous time and cooling rate on the products in the flux method,demonstrating that in this reaction a saturated homogenous solution is obtained upon the melting of the salt and precursor.Afterward,the cooling rate was adjusted to regulate the thickness of the target crystals,leading to the precipitation of 2D non-layered material from the supersaturated solution;by applying this method,the highly crystalline non-layered 2D MoO_(2)flakes with so far the largest lateral size of up to sub-millimeter scale(~464μm)were yielded.Electrical studies have revealed that the 2D MoO_(2)features metallic properties,with an excellent sheet resistance as low as 99Ω·square^(-1 )at room temperature,and exhibits a property of charge density wave in the measurement of resistivity as a function of temperature.
