Construction Of SARS-CoV-2 Replicon By Reverse Genetics

In December,2019,the continuous pandemic of acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious public health crisis and great economic losses worldwide.In order to cope with the new coronavirus,it is urgent to do more research on the biology of SARS-CoV-2,and researchers have developed antiviral agents and established various models.The self-replicating RNA is called replicon,which is a model system used to explore many aspects of the life cycle of RNA virus without producing infectious virus.In this study,we tried to develop a reverse genetic system to generate replication-deficient virus(replicon)as a cell model to provide technical reserves for the study of antiviral drugs or vaccines.SARS-CoV-2 replicon uses the in vitro connection method initiated together with other coronaviruses,and inserts a stable reporter gene expressing green fluorescent protein(EGFP)and nano luciferase(Nanoluc)into the replicon,which can more intuitively evaluate anti-virus preparations and vaccines.The replicon of SARS-CoV-2 has also been used to study the important viral components of SARS-CoV-2.This reverse genetic system provides a key tool for the research of SARS-CoV-2 and the formulation of countermeasures.In this study,the construction process of this reverse genetic system is described.(1)making replicon RNA of SARS-CoV-2.Firstly,the structural transformation of SARS-CoV-2 was completed to reduce the pathogenicity of the reconstructed replicon as much as possible.In the process of transformation,most structural proteins and some non-structural proteins of SARS-CoV-2 were deleted.A green fluorescent protein(EGFP)and nano-luciferase reporter gene were added to facilitate the evaluation of the existence and proliferation of replicons.Because the genome of SARS-CoV-2 is too long,the genome is divided into seven parts(F1-F7)for easy connection,and the F6 fragment with high pathogenicity is deleted.The RNA of wild SARS-CoV-2(BJ01)was extracted,and the wild virus c DNA was obtained by reverse transcription.Primers were designed,viral gene fragments were obtained by PCR and constructed on plasmid vectors PUC57 and PKQLL.The constructed plasmid was expanded and cultured for plasmid extraction.Then,high concentration of virus gene fragments were obtained by enzyme digestion.All the fragments were connected by in vitro linkage,and replicon RNA was obtained by in vitro transcription.(2)Construction of BHK cell line stably expressing N gene.Firstly,the maximum tolerance concentration of G418 antibiotic in BHK cells was confirmed,and the concentration of G418 antibiotic suitable for virus screening was determined.Using SARS-CoV-2c DNA as template,the SARS-CoV-2N gene fragment was obtained by PCR.The n gene was inserted into PCAGGS-G418 plasmid to obtain PCAGGS-G418-N plasmid.PCAGGS-G418-N plasmid was transfected into BHK cells,and the expression of N gene in BHK cells was verified by western blot.After successful verification,plasmid transfection was done again,and then BHK cells were screened with G418 antibiotics.After a period of screening,cell clusters which can be stably inherited in G418 antibiotics were obtained.Single cells were selected for expanded culture,and BHK-N cell line with stable expression of N gene was obtained.(3)Transfect replicon RNA into BHK-N cell line to verify the expression of replicon.Firstly,EGFP plasmid was used to explore the experimental conditions of electrotransformation in order to get higher RNA conversion rate as much as possible.In the course of the experiment,BTX830 and NECO are used.After comparison,NECO has higher efficiency and is selected as the formal experimental instrument.After the completion of electrotransmission,the indexes of cells with and without electrotransmission of replicon RNA were compared.Including the intensity of green fluorescence signal and the comparison of luciferase intensity.To verify the expression of replicon.