Study Of Cloning,Expression And Preparation Of Polyclonal Antibody Of V-ATPase Subunits A,d And C In The Midgut Of Mythimna Separata

The innovation of the target is the basis of the creation of the new pesticide.The study of the drug target can provide the idea for the creation of the new pesticide,and the natural product as the probe is the important way to discover the new target.The Mythimna separata belong to Lepidoptera,Noctuidae,which is a major pest of graminaceous crops.The vacuole type H+-ATPase of the brush border membrane vesicles(BBMV)in the midgut of Mythimna separata can generate energy by the hydrolysis of ATP into form a proton transmembrane gradient to regulate the intracellular pH environment.In recent years,the V-ATPase as a drug target was studied by more and more researchers.In this study,the V-ATPase subunits a,d and C gene in the midgut of Mythimna separata were cloned,prokaryotic expressed and purified,and preparation of polyclonal antibody was studied.The main results are as follows:The full-length cDNA sequence of V-ATPase subunit a(MS-VATPa)gene in the midgut of Mythimna separata was cloned by using RT-PCR and RACE technologies,and the full-length sequence of MS-VATPa gene was 3538 bp,with a 5’UTR of 121 bp and 3’UTR of 905 bp,with a open reading frame(ORF)of 2511 bp,the start codon and termination codon of the ORF is ATG and TAG,encoding a 836 amino acids protein,the predict molecular weight of the protein is 95.75 ku,the predicted isoelectric point(pI)is 6.01.M.sexta exhibits the highest homology of 94%through multiple sequence alignment of amino acid sequences of MS-VATPa from M.separata and other insects.The GenBank accession numbers was KY921845.The full-length cDNA sequence of V-ATPase subunit d(MS-VATPd)gene in the midgut of Mythimna separata was cloned by using RT-PCR and RACE technologies,and the full-length sequence of MS-VATPd gene was 2236 bp,with a 5’UTR of 96 bp and 3’UTR of 1093 bp,with a open reading frame(ORF)of 1047 bp,the start codon and termination codon of the ORF is ATG and TAA,encoding a 384 amino acids protein,the predict molecular weight of the protein is 39.59 ku,the predicted isoelectric point(pI)is 4.93.B.mori exhibits the highest homology of 96%through multiple sequence alignment of amino acid sequences of MS-VATPd from M.separata and other insects.The GenBank accession numbers was KY921847.The full-length cDNA sequence of V-ATPase subunit C(MS-VATPC)gene in the midgut of Mythimna separata was cloned by using RT-PCR and RACE technologies,and the full-length sequence of MS-VATPC gene was 1878 bp,with a 5’UTR of 116 bp and 3’UTR of 611 bp,with a open reading frame(ORF)of 1152 bp,the start codon and termination codon of the ORF is ATG and TAG,encoding a 383 amino acids protein,the predict molecular weight of the protein is 43.75 ku,the predicted isoelectric point(pI)is 8.46.P.xylostella exhibits the highest homology of 88%through multiple sequence alignment of amino acid sequences of MS-VATPC from M.separata and other insects.The GenBank accession numbers was KY921846.The amplified gene fragments of MS-VATPa,MS-VATPd and MS-VATPC were subcloned into prokaryotic expression vector pET22(+)to construct recombinant vectors pET22b-MS-VATPa,pET22b-MS-VATPd,pET22b-MS-VATPC,which were transformed into E.coli BL21(DE3)and induced with IPTG at different concentrations(0.3 mmol/L,0.6 mmol/L,0.9 mmol/L),respectively.The target proteins were purified by using Ni-NTA affinity chromatography,and the purified proteins were verified by SDS-PAGE and Western Blot.The results showed that the MS-VATPa,MS-VATPd and MS-VATPC proteins were successful expressesed and the relative molecular weights of them were about 96.6 ku,40.4 ku and 44.6 ku,respectively.Using the MS-VATPd and MS-VATPC proteins as antigens and immuning the BALB/C rat,respectively.The titer and specificity of antiserums were detected by using ELISA and Weatern Blot,which showed that the preparation of rat anti-MS-VATPd and rat-MS-VATPC polyclonal antibody respectively were 1:4,000 and 1:256,000,and there was a obvious positive stripe with about 44.6 ku which consistented with the theory of relative molecular weight of rat-MS-VATPC polyclonal antibody.The result revealed that the preparation of rat-MS-VATPd polyclonal antibody had comparatively low titer and the preparation of rat-MS-VATPC polyclonal antibody had good specificity and high titer.In summary,the full-length cDNA sequences of MS-VATPa,MS-VATPd and MS-VATPC were successfully cloned,the prokaryotic expression systems of MS-VATPa,MS-VATPd and MS-VATPC gene were respectively constructed,and successfully realized soluble expression of the heterologous gene in the E.coil,and obtained the rat-MS-VATPC polyclonal antibody with good specificity and high titer.The results of this study provide a foundation for further research on the biological functions of MS-VATPa,MS-VATPC and MS-VATPd in insects,particularly the action of them in the process of V0V1 reversible dissociation and assembly of V-ATPase in the M.separata,molecular mechanism of action between drugs and V-ATPase,as well as to the new pesticides screening model of MS-VATPa,MS-VATPC and MS-VATPd,respectively.