Preparation Of Monoclonal Antibody Against EgM123 Protein Of Echinococcus Granulosus

Echinococcosis is one of the zoonoses which seriously affects human and animal health and restricts economic development,and can cause great economic losses to animal husbandry.Due to its high morbidity and mortality rate,most families in western China have been impoverished or returned to poverty due to illness.This disease has been the World Health Organization(WHO)as a statutory report of animal diseases,China’s Min istry of Agriculture will be included in the th ird class animal diseases,the Ministry of Hea lth will be listed as a class C infectious diseases.In thi s study,BALB/ c mice were immunized with EgM123 protein to prepare monoclonal antibody against EgM123 gene,which provided a preliminary basis for the subsequent establishment of echinococcus granu losus detection method.1.p ET28 a and pc DNA3.1/H is-EgM123 were cut by Ecor I and Xho I restriction endonuclease respectively,the target gene was recovered by 1% agar electrophoresis,the recycled gum products were ligated overnight using T4 ligase,the ligand product was transformed into DH5 α competent cells and plasmid was extracted,the plasmid transformed BL21 competent cells were successfully constructed by double enzyme digestion,PCR and sequencing,The expression of EgM123 protein was induced by IPT G,and the protein expression level was explored at different temperatures,times and IPT G concentrations,the EgM123 protein was purified by nickel column affinity chromatography,the purified protein was renaturated by dialysis,and its rea ctivity was detected by Western blot and ELISA,the optimal concentration of EgM123 protein was determined.The results showed that the prokaryotic expression vector p ET28a-EgM123 was successfully constructed by double enzyme digestion,PCR and sequencing,the IPTG induced EgM123 protein expressed by E.coli was inclusion body protein,The optimal induction temperature was 37 ℃,the optimal induction time was 7 h,and the optimal IPTG induced concentration was 0.9 m M,Western blot verified that EgM123 protei n could bind to H is monoclonal antibody and canine EgM123 polyclonal antibody respectively,and the optimal concentration of EgM123 protein was 0.4 μg per well by ELISA.2.BALB/c mice were immunized with EgM123 pr otein,and serum titer of mice was detected by ELISA after three times of immu nization,mice with the highest ti ter were selected,mice spleen cells were fused with myeloma cells using PEG4 000,and subcloned by limited d ilution method,the cells were successfully screened by colchicine and the chromosome karyotypes were determined by Gi emsa stain ing,and antibody subtypes were detected by ELISA.The results showed that the serum titer of the immuni zed mice was 1:25 600,the number of positive hybri doma cells G2 was successfully screened by the fusion of no.1 mous e,the chromosome number was 92 after Giemsa staining.In this study,EgM123 protein with reactivity was successfully prepared,and high antibody level was produced by immunizing BALB/c mice.After cell fusion and screening of positive hybridoma cells,monoclonal antibody was successfully prepared,with heavy chain of antibody subtype Ig G1 and light chain Kappa.