The Study On Subunit Vaccine,Early Diagnosis Method And Allergenicity Based On Seven Recombinant Proteins From Sarcoptes Scabiei

Scabies is a zoonotic ectoparasitic disease caused by Sarcoptes scabiei burrowing into the skin of host.The purpose of this study was to screen out vaccine candidate molecules and a high protection rate vaccine strategy;establish diagnostic method that could diagnose early infestation of S.scabiei;establish method for identifying allergens of scabies mite,and identify some allergens of S.scabiei.This study amplified and expressed scabies mite chitinase-like protein 5(SsCLP5),chitinase-like protein 12(SsCLP12),protein tyrosine kinase(SsPTK),chitin binding protein 1(SsCBP1),chitin binding protein 2(SsCBP2),serine protease inhibitor 3(SsSerpin3),and serine protease inhibitor 4(SsSerpin4).Based on these seven recombinant proteins,a series of studies on subunit vaccine,early diagnosis method and allergenicity were performed.The main work and research results are as follows:1.Screening of candidate antigens of S.scabiei vaccine and optimization of immune programThis study evaluated the immune protective effects of seven recombinant proteins,and optimized the immunization procedures of candidate antigens.The results showed that rSsCLP5,rSsCLP12 and rSsSerpin4 immune groups could induce immune protection in rabbits,while the other four protein immune groups did not have immune protection.The best immunization procedure was 100 μg for single protein immunization,200 μg for mixed protein immunization,and the optimized number of inoculations is twice(2 weeks apart).Studies indicated that rSsCLP5,rSsCLP12 and rSsSerpin4 could be used as candidate antigens for anti-scabies mite vaccine;the optimization of vaccine program would guide for the further research of scabies vaccine.2.Study on the immune protection of single protein vaccine,cocktail vaccine and multi-epitope protein vaccine against S.scabiei The study comprehensively evaluated the immune protective effects of three scabies mite vaccine strategies including single protein vaccine,cocktail vaccine and multi-epitope protein vaccine through vaccine trials.The results showed that the average protection rate of rSsCLP5,rSsCLP12 and rSsSerpin4 was 79.2%,73.9%and 50%,respectively.The average protection rate of cocktail vaccine was 87.5%while that of multi-epitope protein vaccine is the lowest.The results indicated that the cocktail vaccine of the three-protein mixed immunization was a good potential anti-scabies vaccine strategy.3.Characterization of SsPTK and SsCLP5 genes and establishment of early diagnosis method of rSsPTK and rSsCLP5-basedThis study was performed to evaluate the early diagnosis value of rSsPTK and rSsCLP5.Studies showed that rSsPTK and rSsCLP5-based indirect ELIS As showed 95.2%(40/42)and 93.5%(43/46)sensitivity,and 94.1%(48/51)and 90.7%(49/54)specificity,respectively;rSsPTK and rSsCLP5-based indirect ELIS As could diagnose infestation in the early stage(infestation for 1 week)with an accuracy of 100%(24/24).It indicated that rSsPTK and rSsCLP5 could be used to diagnose S.scabiei early infestation as candidate antigens.4.Identification and characterization of potential allergens rSsCLP5,rSsCLP12,rSar s 23 and rSar s 27 from S.scabieiThrough binding test of recombinant proteins with IgE antibody and skin prick test,the sensitization of four recombinant proteins were evaluated and comparatively analyzed.The results showed that these four recombinant proteins all could bind to specific IgE antibodies in mouse sera;and they could also induce skin allergic reactions in rabbits,including rSsCLP5,rSsCLP12 and rSar s 27 induced an immediate allergic reaction,whereas rSar s 23 induced a delayed-type allergic reaction.The study identified four potential allergens of scabies mites,and provided important imformation for studying the secretion and excretion of scabies mites,identification and classification of mite allergens.