Cloning,Prokaryotic Expression And Purification Of V-ATPase Subunit A,B Genes In The Midgut Of Mythimna Separata

The Mythimna separata(Walker)which was belonged to Lepidoptera,Noctuidae,is a major pest of graminaceous crops.The vacuole type(H+)ATP enzyme which was in the Mythimna separata midgut epithelial brush border membrane vesicles(BBMV)can pump the using the energy from the ATP hydrolyzed to maintain the cytoplasmic pH in a relatively stable neutral level.In recent years,the V-ATPase which was because of its important regulatory functions was studied as a drug target by more and more researchers.In this study,V-ATPase A and B subunits gene(vatpa and vatpb)in the Mythimna separata midgut epithelial brush border membrane vesicles were cloned,prokaryotic expression,refolding and purification.The main results are as follows:Total RNA was extracted from the Mythimna separata cells by Trizol.The cDNA was obtained by reverse transcription and amplification of PCR.The vatpa was cloned by RACE.The full-length cDNA of vatpa gene contains 2180 nucleotide and a 1854 bp open reading frame(ORF)which showed that V-ATPase subunit A cDNA contains 1854 nucleotides and will be translated into a 617 amino acids protein,the predict molecular weight(MW)of the protein is 68.21 ku,the predict isoelectric point(pI)is 5.22.M.sexta exhibits the highest homology of 96%through multiple sequence alignment of amino acid sequences of vatpa from Mythimna separata with and other insects.According to V-ATPase subunit A and B genes open reading frame,the specific primers were designed for the amplification of the V-ATPase subunit A and B genes ORF with PCR.The pET22b vector was digested with Xho I and Nde I.PCR products and digested vector fragments were electrophoresis on agarose gel and recovered from the gels.The DNA fragments were linked to the pET-22b vector to construct the prokaryotic expression plasmid named pET22b-vatpa and pET22b-vatpb.Recombinant plasmids pET22b-vatpa and pET22b-vatpb was obtained after being transferred into E.coli BL21(DE3),induced with IPTG after the prokaryotic expression plasmid were successfully constructed.The expression plasmid could be well expressed in Escherichia coli(E.coil).in presence of 0.1 mmol/L,0.5 mmol/L,1 mmol/L IPTG respectively.The recombinant proteins were identified by SDS-PAGE identification which showed that particular 74 ku and 60 ku protein band visualized successfully.The proteins expressed in pET22b Vector were all inclusion bodies existed in the precipitates which was dissolubled by refolding.The supernatant was purified by affinity chromatography with Ni-NTA column,the SDS-PAGE identification was also used to analysis the purified protein,and pure vatpa and vatpb proteins band visualized successfully.In summary,the V-ATPase subunit A and B genes were successfully cloned and expressed in E.coil and the results will facilitate the investigation of crystal structure of V-ATPase subunit A and B,interactions of small-molecule drugs with protein and the creation of environmental friendly pesticides acted on the target of V-ATPase subunit A and B.