Prokaryotic Expression,Purification And Bioinformatics Of Capsid Protein Of Red Clover Necrotic Mosaic Virus

Red clover necrotic mosaic virus?RCNMV?is an icosahedral plant virus including two genomic RNAs that can infect the plants in the Dianthovirus genus in the Tombusviridae family.The advances research indicated that the RCNMV CP has a potential value in the treatment of cancer.The RCNMV CP occurs reversible swelling transition when the p H and divalent ion are changed.The transition results in the opening of pores that allow the small molecules into the cavity,which is open when the divalent ions connected with RCNMV CP were removed by add 200 mMEDTA at pH 8.0;the cavity is closed when put in the divalent ion Ca²⁺.The study obtain the RCNMV cp from the NCBI and then optimized the coding sequence of it according to the E.coli codon preference.The cp was cloned from the synthetic vector pMD18T-cp and then inserted into pET21a-His-MBP-TEV and pMAL-c4x vector to obtain the recombinant vector pET21a-His-MBP-TEV-cp and pMAL-c4x-cp.Then the RCNMV CP was expressed and purified and Bioinformatics analysis of it in order to lay a foundation for the further modification of CP structure and make it become an important carrier of biological target therapy.The results are as follows:?1?According to the sequence of RCNMV cp?Accession number: NC₀03756?from NCBI and optimized the coding sequence of it according to the E.coli codon preference,we synthesized the RCNMV cp and designed the PCR primers which contained restriction enzyme sites and protective bases in their 5' carbon terminal.The target cp was cloned by PCR.?2?Constructing the recombinant expression vectors pET21a-HMT-cp and pMAL-c4x-cp,and then the recombinant vector was transformed into E.coli BL21?DE3?p Lys S and Rosetta?DE3?and was expressed under the the induction of isopropyl-?-D-thiogalactoside?IPTG?.The results showed that pET21a-HMT-cp had been expressed in BL21?DE3? pLysS and some of them were soluble.However, pMAL-c4x-cp in Rosetta?DE3?were expressed in the form of insoluble.?3?Nickel affinity chromatography column?Ni-NTA?as well as maltose chromatography column?Amylose?was used for purification of fusion protein and the purified fusion protein was digested by protease TEV at 30? with 2 h,4 h and 6 h respectively to remove MBP tag and His tag.The SDS-PAGE gel electrophoresis results showed that the target protein successfully expressed,which was obtained by 4 h digestion.?4?Bioinformatic analysis of RCNMV CP suggests that signal peptide and rare codons did not exist.The secondary structure was given priority to random coil.Most of CP was secreted outside of cells and tertiary structure of CP was composed of three subunits, with each made up of ?-helix and ?-sheet.