Preparation Of Monoclonal Antibody Against Brucella And Preliminary Identification Of The Antigen

Brucellosis is a crucial zoonosis caused by Brucella,which is a serious threat to animal husbandry and human health.Over the past decade,brucellosis is a comprehensive epidemic in our country,and it has bring great economic loss,to carry out the prevention and control of brucellosis without delay.Accurate detection of brucellosis is an important part of prevention and control,and serological detection is the most commonly used detection method of brucellosis.Enzyme-Linked Immunosorbent Assay(ELISA)has the advantages of high efficiency,sensitivity and specificity,so it has become one of the main methods of detection of brucellosis.Development and application of the competitive ELISA technique(cELISA)based on monoclonal antibody,which has higher sensitivity and better specificity.The cELISA is widely used in nowadays,as it can be used for the detection of brucellosis in different animals.However,specific monoclonal antibodies with good specificity and high sensitivity to Brucella are prerequisites for developing competitive ELISA kits.In this study,BALB/c mice were immunized with inactivated bacteria Brucella suis 1330 as antigen.After immunization program,spleen cells of the best immune effect mice were collected,and fused with SP2/0 cells in 5: 1 ratio.The indirect ELISA(iELISA)method was established by using different strains of bacterial,including B.suis 1330,B.abortus 2308,B.melitensis 16 M,B.melitensis rough mutant57(RM57),Escherichia coli O:157,Yesinia enterocolitica O:9 as screening antigen.After three times subclones,a hybridoma cell line 6E4 that secreting the monoclonal antibody to Brucella was obtained.The monoclonal antibody was specifically reacted with smooth Brucella: B.suis 1330,B.abortus 2308,B.melitensis 16 M,but has no cross reaction with RM57,as well as Escherichia coli O:157 and Yesinia enterocolitica O:9.The method of immunizing proteome was further applied to screen the antigen and its epitope recognized by the monoclonal antibody.After the two-dimensional electrophoresis of Brucella protein,the monoclonal antibody 6E4 was used to Western blot,and differential protein analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS).The result show that seven Brucella proteins may react with monoclonal antibody 6E4,and labeled L11,L12,L13,L14,K11,K12,K13.According to the reference sequence published in Genbank,seven primers of protein coding genes were designed and the DNA fragments of corresponding genes were obtained by PCR.The PCR products were cloned into the expression vector pET-28 a and transformed into E.coli BL21(DE3)for expression and purification.The purified protein was identified by Western-blot using monoclonal antibody 6E4.The results showed that L12,L13 and L14 had no specific reaction with monoclonal antibody 6E4.K11,K12,K13 and L11 reacted with monoclonal antibody 6E4,and K11 had the strongest reactivity.Four positive proteins were identified as suspicious antigens and need further validation.In this study,we screened a specific monoclonal antibody against Brucella,and predicted and expressed seven protein antigens.It was confirmed that four protein antigens had specific reaction with monoclonal antibody.Completed work has laid the foundation for the development of a competitive ELISA kit for Brucella with good specificity and high sensitivity.