Descriptive Preparation And Preliminary Application Of Novel Coronavirus Pseudovirus System

ObjectiveSevere acute respiratory syndrome coronavirus 2(SARS-Co V-2),which periodically emerges around the world,poses a threat to human health.SARSCo V-2,also known as Novel Coronavirus,is highly transmissible and highly pathogenic.In this study,by constructing a VSVΔG*-S pseudovirus carrying the S protein of SARS-Co V-2,we can also conduct research on SARS-Co V-2 in a biosafety secondary facility.At the same time,the prepared pseudovirus was used to evaluate the neutralizing effect of serum antibodies and to initially explore the inhibitory effect of polypeptide drugs on viral infection,so as to provide technical support for the further development of treatment plans and preventive drugs for novel coronavirus pneumonia.Methods1.Construction of recombinant plasmids: The DNA sequence of the SARSCo V-2 spike protein(S protein)was synthesized,and the sequence was amplified by PCR and then connected to the pc DNA3.1(+)vector to construct the pc DNA3.1(+)-S recombinant plasmid.The successfully constructed plasmid was verified by double digestion with Hind III and Xho I and sequencing.2.Expression of S protein: The pc DNA3.1(+)-S recombinant plasmid was transfected into Vero cells by PEI transfection reagent,and the expression of S protein in the cells was detected by Western blotting and immunofluorescence experiments.3.Preparation of VSVΔG*-S pseudovirus: The pc DNA3.1(+)-S recombinant plasmid was transfected into Vero cells by PEI transfection reagent,and then infected with VSVΔG*-G virus.After 24 hours,the supernatant was collected,and the VSVΔG*-S pseudovirus was obtained after centrifugal filtration.Pseudoviruses were aliquoted and stored in a-80°C freezer.4.Determination of pseudovirus titers titers: Pseudovirus titers were calculated using the fluorescence foci method.Vero cells were seeded in a 24-well plate,and the pseudovirus was diluted 10-fold(10-1~10-7)to infect the cells.The expression of GFP was observed under an inverted fluorescence microscope,the results were recorded and the virus titer was calculated.5.Pseudovirus Neutralization Test to Detect the Neutralization Level of Serum Antibodies: Collect the serum of volunteers of different ages who have been vaccinated with three doses of the new crown vaccine,incubate the serum with the VSVΔG*-S pseudovirus and infect Vero cells,initially observe the fluorescence changes under an inverted fluorescence microscope,and detect the expression of green fluorescent protein(GFP)by flow cytometry.6.Detection of peptides preventing pseudovirus from entering cells: After co-incubating diluted peptides with VSVΔG*-S pseudovirus,vero cells were infected.The fluorescence changes were initially observed under an inverted fluorescence microscope,and then the inhibitory effect of the peptide on the pseudovirus adsorption was detected by flow cytometry.Results1.Construction of recombinant plasmids: After agarose gel electrophoresis and double-enzyme digestion verification,the pc DNA3.1(+)-S recombinant plasmid was successfully constructed.The sequencing results also showed that the recombinant plasmid was constructed successfully.2.Expression of S protein: It was verified by Western blotting and immunofluorescence experiments that S protein could be successfully expressed in Vero cells.3.Preparation of VSVΔG*-S pseudovirus: The expression of GFP in cells infected with VSVΔG*-S pseudovirus was observed under an inverted fluorescence microscope,indicating that the VSVΔG*-S pseudovirus was successfully prepared.4.Determination of pseudovirus titers: The titer of pseudovirus was2.66X105 FFU/ml measured by fluorescence foci method.5.Pseudovirus Neutralization Test to Detect the Neutralization Level of Serum Antibodies: The neutralization test showed that,compared with the control group without serum,the infectious activity of the pseudoviruses coincubated with serum was significantly inhibited.6.Detection of peptides preventing pseudovirus from entering cells: The results of flow cytometry showed that the number of GFP-carrying cells decreased after co-incubation of the peptide and pseudovirus,suggesting that the infection activity of the pseudovirus was inhibited.Conclusions1.The pc DNA3.1(+)-S recombinant plasmid was successfully constructed.2.The VSVΔG*-S pseudovirus was successfully prepared.3.Pseudoviruses can be used in neutralization assays to evaluate the neutralizing activity of serum antibodies.4.Using the pseudovirus system,it can be detected that the polypeptide designed to target the S protein can bind to the blocking virus and the receptor,thereby inhibiting the adsorption of the virus,preventing the virus from entering the cell,and playing an antiviral role.