| Objective:To construct a resource library of Shanxi wild Buthus martensii Karsch venom peptides by transcriptomics,proteomics,molecular sieve,high performance liquid chromatography and mass spectrometry.To find new BmK sodium channel toxins by screening the library.To study characterization and activity of the new BmK sodium channel toxin BmKNaTx12.Methods:(1)The transcriptomics and cDNA library of Shanxi wild Buthus martensii Karsch was constructed,the homologous genes which were obtained by recognizing,splicing and comparing unigenes went to Blast for biological functions and screened out possible genes for new toxins.(2)Proteomics of Shanxi wild Buthus martensii Karsch was constructed.Venom peptides were marked and quantified by iTRAQ,and identified by SCX separation,HPLC and MS.(3)The natural venom of BmK was separated by Sephadex G50 gel filtration chromatography,HPLC,and combined electrophysical activity screening.The result showed that BmKNaTxl2 is a new sodium channel toxin from BmK.(4)BmKNaTxl2 showed high homology similarity with Cn12 from Mexican Centruroides noxius scorpion by sequence alignment.The predicted structure of BmKNaTxl2 was obtained by homologous model construction and molecular dynamics optimization.A new recombinant expression system of BmKNaTxl2 was constructed.rBmKNaTx12 was expressed in HEK293 cells,obtained by protein A affinity chromatography,and identified by SDS-PAGE and HPLC.(5)The effects of BmKNaTxl2 on hNavl.7 and rNavl.8 were tested by whole-cell patch-clamp assay.Results:(1)Capacity of BmK cDNA library was1.6×106 cfu/ml,while recombination rate was 93.75%.The average length of the inserted sequence was more than 750bp.36665 unigenes were obtained after splicing original sequences,2231 homologous genes were screened by NCBI database,and more than 50%genes were related to toxins.The result of function annotation of transcriptomics showed 8%of the sequences were suspected to be new Na+ channel toxins.(2)Mascot analysis of proteomics identified 245 proteins.(3)49 single component proteins from BmK were identified by gel filtration chromatography and HPLC.It turned out that 19 new toxin peptides were identified,and 9 of which seems to be sodium channel toxins.According to results of transcriptomics and proteomics,BmKNaTx12 was selected and classified to be a new sodium channel toxin.(4)The homology similarity of BmKNaTxl2 to Cn12 was 35%,the structure of BmKNaTx12 was simulated by the structure of Cn12.The recombinant expression vector of BmKNaTxl2 was successfully constructed,and BmKNaTxl2 was successfully expressed in HEK293 cells.Recombinant protein was purified by protein A affinity purification column to 30ml solution with concentration of 0.12mg/ml.SDS-PAGE gel electrophoresis showed that the size of the film was about 40kD,and result of high performance liquid phase showed high purity of BmKNaTx12.(5)The whole-cell patch-clamp assay results suggested that 1?mol/L BmKNaTxl2 could increase 20%hNav1.7 peak current,while 1?mol/L BmKNaTxl2 had no effect on rNavl.8 current,it showed that recombinant BmKNaTx12 can significantly activate Nav1.7 current,which may have potential to cause pain.Conclusion:A resource library of Shanxi wild Buthus martensii Karsch venom peptides was constructed by transcriptomics,proteomics,molecular sieve,HPLC and MS.A few of new Na+ channel toxins,represented by BmKNaTx12,were found and identified.Recombinant BmKNaTxl2 was successfully expressed,and significantly increased hNav1.7 peak current.Take BmKNaTx12 as a template,the strategy of finding new Na+ channel toxins was established,it has laid a foundation for the subsequent study of biological function of BmK Na+ channel toxins. |
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