Screening And Identification Of Deinagkistrodon Acutus Protective Antigen Based On Antigenome Technology

Almost3000species of snakes are known and widely distributed the most areas of theworld. But only15%snake species are venomous and potentially dangerous to human.Snakebite envenoming mainly occur in villages of tropics and subtropical zone. Since thereare many countries where this disease is not appropriately reported, the actual incidence ofsnakebite envenoming world wide and its associated mortality are difficult to estimate. Arecently published study estimated a total of2.5million envenomings and over125,000deaths per year. In2009, the world health organization (WHO) first recognized that snakeenvenoming constitutes a highly relevant public health issue, although it has beensystematically neglected by health authorities in many parts of the world.Deinagkistrodon acutus, differing from other species, only located in Southern China.Several components have been reported including metalloproteinase, phospholipase A2,serine proteases, C-type lectin-like protein. Deinagkistrodon acutus venom is typicalHemotoxin, responding for Systemic hemorrhage and local tissue necrosis. It brings seriousdifficulties for clinical treating victim by Deinagkistrodon acutus bite.Phage random peptide display is a new technique for studying the epitope of simulatedantigens. For the past few years, this technique has been successfully used to mimotopesscreening and inducing humoral immune response in animals. A certain length of thepeptide is expressed as fusion protein displayed on the phage surface. By immunoscreening,the specific phage clone was obtained. Then the peptide can be sequenced to acquire thecorresponding information, such as the structure and the function. By now, the techniquehas been widespread used for determining the antigen epitope, Screening of small molecularpeptide, vaccine development and molecular diagnosis.Objective To purify specific polyclonal antibody against Deinagkistrodon acutusvenoms and detect the biological activity of specific polyclonal antibodies. Based on antigenome technology, the specific polyclonal antibodies are treated as target protein forscreening and identifing of the Deinagkistrodon acutus protective antigen. These selectedantigens are recombined expressed with genetic engineering technology. The immunizingprotection of recombinant peptide is evaluated by animal experiment.Methods: This study is consisted of two parts.Part one: New Zealand white rabbits were immuned with Deingagkistrodon acutusvenom. The IgG fraction was purified by affinity chromatography with the Econo-PacProtein A Kit. Specific antibody was purified by immuno-affinity chromatography withCNBr activated sepharose4B.Then the polyclonal antibody was tested by Elisa assay andWestern blotting analysis to evaluate its immunocompetence. Antibody biological activityof the polyclonal antibody was evaluated by Lethality neutralization assay andneutralization of venom hemorrhagic activity.Part two: Polystyrene micro-ELISA plates were coated with the specific polyclonalantibody against Deinagkistrodon acutus venoms. After three rounds biopanning, thepositive clones were obtained. Positive phage clones were tested for their specific binding toSba-pab by indirect ELISA and Western blot. The single strand phage DNAs of the positivephage clones were extracted from the phage particles and sequenced with the phage flank96-primer. The phage peptide sequences were deduced from the DNA sequences. Thesepeptides were synthesized by genetic engineering technology. BALB/c mice wereimmunized recombinational peptide. Then the mouse serum was collected and antibody titerwas tested by indirect ELISA. The capacity of sera from the immunized animals toneutralize the hemorrhagic and lethal effects of various venoms was evaluated.Results:1) The specific polyclonal antibody against Deinagkistrodon acutus venoms wasprepared successfully. The ELISA test showed its antibody titers higher than3200. Westernblotting indicated that the polyclonal antibody can specific recognize the most protein ofDeinagkistrodon acutus venoms. Neutralization of venom hemorrhagic activitydemonstrated the polyclonal antibody can effectively neutralize intradermal hemorrhageinduced by Deinagkistrodon acutus venoms. In the Lethality neutralization assay, thepolyclone antibodies can provide100%and54%protection against2LD50and4LD50doseDeinagkistrodon acutus venoms respectively. 2) In this work, five epitopes were screened out. To acquire recombinant peptides,these epitopes were artificially synthesized by tandem expression method. It is confirmedthat the recombinant peptide can stimulate the production of antibodies in mice that can berecognized by recombinant peptide and Deinagkistrodon acutus venoms. The antibody titeris higher than1/64000and1/8000recognized by recombinant peptide and Deinagkistrodonacutus venoms respectively. The anti-D588antiserum resulted in a100%reduction in themean area of hemorrhage induced by2MHD D. acutus venom and82%reduction by4MHD D. acutus venom.Conclusion: In this research, five epitopes were successfully obtained and theseepitopes have immunizing protection against Deinagkistrodon acutus venoms. Our researchestablished a foundation for the further research and development of new type antivenoms.