Study On The Construction,Expression And The Immunological Characteristics Of Echinococcus Granulosus Recombinant BCG-EgG1Y162 Vaccine

Objective:To construct and express the Echinococcus granulosus recombinant plasmid BCG-EgG1Y162,analysis its protein expression and investigate its immunological characteristics.Methods:Amplified the antigen gene of EgG1Y162 by PCR method.This PCR product was cloned into Escherichiacoli-Mycobacterium shuttle plasmid pMV361 to construct the recombinant plasmid pMV361-EgG1Y162.The recombinant plasmid and the empty plasmid were electroporated into BCG respectively to construct rBCG-EgG1Y162,BCG-pMV361;The rBCG bearing rBCG-EgGlY162was induced by high temperature of 45℃;and expressed protein was identified by SDS-PAGE and Western Blotting;inoculated mice with BCG-pMV361,rBCG-EgG1Y162,Physiological saline respectively.The proliferative reaction of splenic lymphocyte invitro were tested by cck-8.Results:It is demonstrated that the 360bp EgG1Y162 gene was successfully amplified by cloned into pMV361 by restriction analysis and transformed into BCG.71kDa specifical protein identified by SDA-PAGE and western Blotting,Western blot analysis showed that the expression of rBCG-EgG1Y162 in relative molecular mass(Mr)of about 71KDA has obvious target protein strip,and the rBCG-EgG1Y162 can specifically recognized the polyclonal serum which prepared by immunizing rabbit with recombinant EgG1Y162 protein,Echinococcus granulosus canine serum respectively.The splenic lymphocyte proliferate activity in rBCG-EgG1Y162 group in vitro was significantly higher then those BCG-pMV361 and Physiological saline group;Immunogenicity tests showed that immunization for 6 weeks with rBCG-EgG1Y162 in mice,the IgG1,IgG2a,IgG2b antibody subclass levels higher than the control group,there was a significant difference compared with BCG-pMV361 group and saline group(P<0.05).Conclusion:Successfully constructed the recombinant plasmid BCG-EgGlY162 of Echinococcus granulosus,it can stability and highly expressed the target protein;after immunization with rBCG-EgG1Y162 in mice,its organism generated high levels of IgG1,IgG2a,IgG2b antibody,can induced cell immunity and humoral immunity reaction,illustrated that rBCG-EgG1Y162 has better immunogenicity.This research lay the theoretical and experimental basis for the further development of hydatid disease vaccine.