Preparation And Preliminary Application Of Monoclonal Antibodies Against Salmonella Pullorum IpaJ Protein

Salmonella Pullorum(S.Pullorum)is a host-specific pathogen,which mainly infects within 21 day-old chicks and causes acuses bacteremia and white dysentery with extremely high mortality.Although infected adult chickens may have no clinical symptom,they can be carriers combined with weight loss and laying drop.S.Pullorum contains many pathogenic genes,of which the ipaJ gene is a newly detected virulence-associated one.It has been confirmed that IpaJ protein involved in the early stage of S.Pullorum infection process.Additionally,ipaJ is a unique gene,which is widely distributed in S.Pullorum isolates.However,the gene was not detected in other closely related Salmonella serotypes,such as Salmonella Gallinarum and Salmonella Enteritidis.In this study,the ipaJ gene of S.Pullorum was cloned into prokaryotic plasmids and expressed in E.coli,the monoclonal antibodies against IpaJ were then prepared and used to establish an indirect ELISA method to detect pullorum disease.All of these work provided important biological materials for study on pathogenesis of S.Pullorum and pullorum disease diagnosis.1.Cloning and expression of ipaJ gene from Salmonella PullorumThe ipaJ gene was cloned by PCR using S.Pullorum C79-13 and Shigella flexneri SNI genome as templates.The ipaJ gene were cloned into the prokaryotic expression vector pColdI and pMAL-c5X.Digestion of recombinant plasmids by restriction enzymes and DNA sequencing analysis demonstrated that ipaJ was successfully cloned into the prokaryotic expression vectors without mutation.These recombinant plasmids were named pColdI-SP-ipaJ(S.Pullorum),pMAL-c5X-SP-ipaJ(S.Pullorum),and pMAL-c5X-SF-ipaJ(Shigella flexneri).The recombinant prokaryotic plasmids were then transformed into the corresponding host cells BL21(DE3)and ER2523 by heat shock.After induction at 0.5 mM or 0.3 mM IPTG,the expressed proteins were analyzed by SDS-PAGE and Western Blot.The results showed that the recombinant protein bands were 31.8 kDa(rHis-SP-IpaJ),73.5 kDa(rMBP-SP-IpaJ),and 71.3 kDa(rMBP-SF-IpaJ).The rHis-SP-IpaJ was recovered from SDS-PAGE gel,and immunoaffinity column chromatography technique.Western Blot confirmed that the purified target proteins have good immunoreactivity,indicating that the recombinant proteins rHis-SP-IpaJ,rMBP-SP-IpaJ,and rMBP-SF-IpaJ were successfully expressed and purified for further study.2.Preparation of monoclonal antibodies against IpaJThe recombinant gel-purified recombinant protein rHis-SP-IpaJ was used as immunogen to immunize subcutaneouly 6-8 week-old BALB/c mice.After immunized three times,cell fusion was performed using lymphocyte hybridoma technique.The purified recombinant protein rMBP-SP-IpaJ was used as detecting antigen,and the supernatant of positive hybridoma clones were screened by indirect ELISA.After three times of detection,eight positive hybridoma cell lines stably secreting IpaJ monoclonal antibodies against IpaJ named 1C3,1F3,2D9,3B5,3D8,4D5,4G6,and 5D1 were obtained.The immunoglobulin subclass identification results showed that 1C3,2D9,4G6 and 5D1 were IgGl,3B5 and 4D5 were IgM,1F3 and 3D8 were IgG2b.The in vitro ELISA method was used to determine the titers of cell supernatants.1C3,2D9 and 3B5 were 200,1F3 and 3D8 were 1600,4D5,4G6 and 5D1 were 6400;The result of ascites titers showed that 1C3 was 2000,1F3 was 256000,2D9 was 1000,3B5 was 4000,3D8 and 4D5 were all 128000,4G6 was 2048000,and 5D1 was 4096000.ELISA and Western Blot showed that all of eight monoclonal antibodies had good reactivity with the recombinant proteins rHis-SP-IpaJ,rMBP-SP-IpaJ and rMBP-SF-IpaJ,However,there was no reaction with the control BL21(DE3)-pColdI and ER2523-pMAL-c5X empty vector bacteria.The results of specific identification showed that eight monoclonal antibodies only reacted with the corresponding recombinant proteins,and with no cross reaction with other recombinant fusion proteins.3.Preliminary application of monoclonal antibody against IpaJThe 4G6 labelled with HRP was used to analyze the expression of IpaJ from S.Pullorum cultivated in vitro and a competition ELISA assay was established using the recombinant protein rMBP-SP-IpaJ as the detecting antigen.The results of Western Blot revealed that IpaJ protein was expressed by S.Pullorum cultured in LB medium.When the working concentration of the serum was 1:10,competitive ELISA square array determined the concentration of coated antigen and enzyme competition antibody 4G6,and optimal concentration of coated antigen was 0.5 ?g/mL,best enzyme competition antibody was 1:16000.The gradient experiments determined the best working concentration of serum was 1:2.The ROC curve was plotted,and sensitivity was 0.906,specificity was 0.762,the Cut-off value was determined to be 40.5.Statistical analysis was performed at p<0.05.Four out of 200 chicken sera samples from one farm were detected to be positive in antibodies against IpaJ using the competitive ELISA method.This result indicated the established competitive ELISA could be applied in the diagnosis of pullorum disease in chicken farms.