Construction Of Clonal Expression Vectors Of Non-structural Proteins Nsp10 And Nsp16 Genes Of SARS-CoV-2 And Preparation Of Substrates

Object Novel coronavirus(Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)belongs to the coronavirus,a righteous single-stranded RNA virus with an envelope that causes novel coronavirus pneumonia(Corona Virus Disease 2019,COVID-19).In this study,the gene sequences of SARS-CoV-2 nonstructural proteins Nsp10 and Nsp16 were cloned,the two proteins were expressed by a prokaryotic expression vector system,and Nsp10 and Nsp16 and Nsp10/Nsp16 fusion proteins were successfully prepared,and the RNAs were capped by in vitro transcription to obtain Nsp10 and Nsp16 functional substrates used for the assay.Methods(1)Primers were designed based on the gene sequences of SARS-CoV-2 nonstructural protein Nsp10(NCBI: YP＿009725306.1)and Nsp16(NCBI: YP＿009725311.1)published by Genebank,and the Nsp10 and Nsp16 genes were amplified by PCR from SARS-CoV-2 The Nsp10 and Nsp16 genes were amplified from the c DNA fragment of SARS-CoV-2 by PCR,and the target genes were fused with His gene tags and then ligated into the prokaryotic expression vectors PET15 b and PET30 a using restriction endonuclease double digestion to construct the recombinant plasmids.The recombinant plasmids were subjected to PCR and enzyme digestion reactions for initial identification,and the initially identified plasmids were sequenced.If the sequences of cloned Nsp10 and Nsp16 genes are identical to the published genes,it indicates that the prokaryotic expression vector was successfully constructed.(2)Optimization of recombinant plasmids PET15b-Nsp10 and PET30a-Nsp16 for induction of expression.The successfully constructed prokaryotic expression recombinant plasmids PET15b-Nsp10 and PET30a-Nsp16 were transformed into E.coli receptor cells BL21(DE3),identified by PCR of bacterial broth,and optimized by induction of time and IPTG concentration for protein expression.(3)SDS-PAGE electrophoresis and protein immunoblotting(Western Blot,Wb)were used to verify whether the target protein was being successfully expressed.SDS-PAGE electrophoresis and Wb were used to verify whether Nsp10 and Nsp16 proteins existed in soluble form.To obtain Nsp10 and Nsp16 proteins with high purity,the expression and purification conditions of nonstructural proteins Nsp10 and Nsp16 were optimized,and nickel chromatography columns were used for nonstructural protein Nsp10,Nsp16 and Nsp10/Nsp16 fusion proteins were purified.(4)Preparation of RNA plus cap substrates.The c DNA fragment of UTR of SARS-CoV-2 was cloned by KOD enzyme,and RNA was obtained by in vitro transcription using an in vitro transcription kit,and RNA was capped by Capped RNA Synthesis capping system to obtain RNA capping substrate.Results(1)The replicon of SARS-CoV-2 coronavirus nonstructural protein Nsp10 and PET15 b plasmid were ligated by restriction endonuclease Bam HI and Xho I,and the recombinant plasmid was named PET15b-Nsp10.The replicon of SARS-CoV-2 coronavirus nonstructural protein Nsp16 and PET30 a plasmid were ligated by restriction endonuclease Nde I and Xho I,and the recombinant plasmid was named PET30a-Nsp16.The recombinant plasmid was named PET30a-Nsp16,and the sequencing results showed that the recombinant plasmids PET15b-Nsp10 and PET30a-Nsp16 were successfully constructed.(2)The recombinant plasmids PET15b-Nsp10 and PET30a-Nsp16 were transformed into E.coli by blue-white screening after cloning the correctly sequenced PET15b-Nsp10 and PET30a-Nsp16 expression plasmids,planted on LB culture plates with resistance,and incubated for 12-14 hours at 37°C.After picking the monoclonal strain by optimizing the culture conditions The PET15b-Nsp10 and PET30a-Nsp16 recombinant plasmids were selected for expression of a large number of target proteins by incubation with 0.1 m M IPTG for 16 h.(3)Nsp10 and Nsp16 proteins were found to be abundantly expressed and present in soluble form by SDS-PAGE electrophoresis and Wb.The purification conditions were optimized for the non-structural proteins Nsp10 and Nsp16 and purified by nickel chromatography column,and the purified Nsp10 and Nsp16 proteins were obtained,and the bacteria were mixed for Nsp10/Nsp16 fusion protein purification.(4)The RNA of the UTR of SARS-CoV-2 coronavirus was transcribed in vitro and the RNA was capped by the Capped RNA Synthesis capping system to obtain the RNA-acting substrate.Conclusion(1)The SARS-CoV-2 coronavirus Nsp10 and Nsp16 expression vectors PET15b-Nsp10 and PET30a-Nsp16 were successfully constructed.(2)Successfully expressed PET15b-Nsp10 and PET30a-Nsp16 recombinant plasmids,successfully optimized conditions and expressed Nsp10 and Nsp16 proteins.(3)Successfully purified Nsp10 and Nsp16 proteins and Nsp10/Nsp16 fusion protein by nickel chromatography column,which provides the basis for in vitro function study.(4)The substrate RNA was successfully obtained by in vitro transcription for capping,providing a substrate for methylation function assay.