Multi-omics Analysis And Antibody Preparation Of Snake Venom Components From The Sea Snake

Snakebites globally remain a common health problem.The main cause of the injury or mortality associated with snakebites is snake venom,which is a complex mixture of enzymes as well as other proteins and polypeptides secreted by specialized venom glands.Identifying the various toxins found in snake venom will help doctors treat victims bitten by snakes.Antisera are currently the most effective medication for the treatment of snakebites.However,there were relatively few studies on preparation of antisera targeting sea snakes,especially common sea snakes in China.This study included three pieces of major work:The first piece studied the composition and content of proteome of Hydrophis cyanocinctus venom by RP-HPLC,SDS-PAGE and LC-MS.The second piece was based on high-throughput sequencing technology to sequence the transcriptome of venom gland from H.cyanocinctus based on high-throughput sequencing technology.Determining the characterization of the venom-gland transcriptome.The third piece studied preparation of mono-and bi-specific antisera raised in rabbits against venoms of H.cyanocinctus and Hydrophis curtus.And detecting the neutralizing effect between antivenoms and snake venoms,the cross-treatment of snake wound model animals and the feasibility of antisera in clinical treatment.The main results of this study as follows:1.Identification of proteome characterization of snake venom from H.cyanocinctus.Twenty liquid component peaks were separated from the crude venom of the sea snake venom by reversed-phase liquid chromatography.Twenty-six protein bands were finally separated by SDS-PAGE for mass spectrometry identification.The proteomic analysis results showed that H.cyanocinctus venom was mainly composed of three toxin families,including phospholipase A₂(40.1%),three-finger toxin(58.1%)and cysteine-rich secretory protein(1.8%).In addition,alkaline phospholipase A₂and long-chain neurotoxin accounted for 22.6%and 38.9%of the total,respectively,which were the main components of the phospholipase A₂and three-finger toxin family.In general,the liquid phase peaks 1-9 were mainly three-finger toxin,accounting for 54.9%of the total protein of snake venom.The liquid phase peaks10-19 were mainly phospholipase A₂,accounting for 40.1%of the total,and the peaks 10,12-14 contained a small amount of three-finger toxin(3.2%).The peak 20only contained cysteine-rich secreted protein.2.Identification of transcriptome characterization of venom gland from H.cyanocinctus.The toxin group was encoded by only 59 transcripts,with the FPKM abundance,non-toxin group and unidentified group accounting for 85.7%,13.4%and 0.9%of the H.cyanocinctus venom gland transcriptome,respectively.Fifty-nine toxin transcripts were belonging to 24 toxin families,including 46 complete and 13 incomplete CDS sequences in transcripts.Three-finger toxin and phospholipase A₂were the main components of toxin,which accounted for 69.8%and 29.0%of total toxin transcript FPKM.The rest twenty-two toxin families only accounted for 1.3%.Twenty-two toxin transcripts with complete CDS sequences were selected as objects for evolutionary selection analysis,and codeml test was used to detect the positive selection effect of each toxin.The results showed that three-finger toxin(3-FTx),cysteine-rich venom protein(CRISP),cysteine inhibitor(Cystatin),dipeptidylpeptidase IV(DPP IV),L-amino acid oxidase(LAAO),nerve growth factor(NGF),phospholipase A₂(PLA₂),phospholipase B(PLB),snake venom metalloproteinase(SVMP)and venom factor(VF)showed significant positive selection.5'nucleotidase(5'NT),acid phosphomonoesterase(AP),hyaluronidase(HA),phospholipase A₂inhibitor(PLA₂inhibitor),glutaminyl-peptide cyclotransferase(QC)and vascular endothelial growth factor(VEGF)showed no positive selection.One of three C-type lectin(CTL)transcripts showed significant positive selection.One of two Kunitz transcripts showed significant positive selection.There were 14 transcripts belonging to ten toxin families with only one homologous sequence,including three-finger toxin,5'nucleotidase,acid phosphomonoesterase,CREGF,Cystatin,phosphodiesterase,phospholipase A₂inhibitor,snake venom metalloproteinase,metalloproteinase inhibitor and Waprin.And yn00 test was used to detect the positive selection effect of each toxin.Three-finger toxin(three transcripts)and snake venom metalloproteinase(one transcript)showed significant positive selection,while the rest of transcripts showed no positive selection.3.Preparation and detection of sea snake antisera.The results of enzyme-linked immunosorbent assay showed that the rabbit antisera generally showed clearly detectable immunological cross-reactions after the third immunization and indicated that the strength of cross-reactions increased with an increase in the immunizing dose.Proteins within the H.cyanocinctus and H.curtus venoms showed similar profiles and were mainly concentrated in the low-molecular-weight region(8-25 k Da).Western blotting results revealed that the bands of these low-molecular weight proteins were dense and showed strong immunogenicity.Although we detected comparatively few bands of the high-molecular-weight proteins,these also showed strong immunogenicity.Our results indicate that both mono-and bi-specific antisera both can neutralize H.cyanocinctus and H.curtus venoms,and in this regard,the mono-specific H.curtus and bi-specific antiserum were found to be superior to the H.cyanocinctus antiserum.