Development And Identification Of Monoclonal Antibodies Against Salmonella Pullorum PagC Protein

Salmonella are gram-negative bacilli which can live in the intestines of both humans and animals.Infected animals will lead to sepsis,gastroenteritis,and local inflammation.Infected people will cause diseases such as typhoid,paratyphoid,acute gastroenteritis and sepsis.It hinders the development of breeding industry and public health in great degree.Particularly,it has caused huge economic losses to the poultry industry in China.Purification of pullorosis is a key development projects in China and eveloping an accurate detection is the premise of this work.So far,the most widely used methods is serum-plate agglutination test,for it is easy to operate and low in cost.But its sensitivity and specificity are poor.Therefore,it is urgent to develop new detection method.Blocking ELISA using a monoclonal antibody has been widely used in the diagnosis of various diseases due to its advantages of high sensitivity,rapidity,simplicity and high specificity.In this study,specific monoclonal antibodies against Salmonella were prepared to help the control of salmonellosis.The contents of research are as follows:1.Development of the monoclonal antibodies against Salmonella PagC proteinAmino acid sequence alignment showed that PagC protein is widely distributed in Salmonella enterica subsp.enterica and less than 65%homology with those of other common non-Salmonella bacteria.To express recombinant protein PagC of Salmonella typhimurium using prokaryotic expression system.The purified PagC was used to immunize BALB/c mice and the spleen cells of mice were fused with myeloma cells.Six monoclonal antibodties(mAbs),named A?B?C?D?I and J,were gotten after subcloning and indirect ELISA.The indirect ELISA titers in supernatant were 1:800-1:1600.Western blot analysis revealed that six mAbs were highly specific to rPagC,indicating that the monoclonal antibody against rPagC of S.Typhimurium was successfully prepared.2.Identification of B cell epitope of monoclonal antibodies against P1 or P2The sequences of S.Typhimurium PagC protein were compared in NCBI.Finally,we got two conserved amino acid sequences,CDRQASGSVEPEGIH named P1 and CFKEHSTQDGDSFNKISSRKTGFA named P2,which are widely distributed in Salmonella enterica subsp.enterica but not distributed in non-Salmonella.They were synthesized artificially,then coupling linked with keyhold limpet hemocyanin as complete antigen,called KLH-Pl and KLH-P2.After using Ellman kit to ensure the peptide coupling is successful,we use KLH-P1 and KLH-P2 to analyze B cell epitope of mAbs.The results showed that mAb J could bind to P1,indicating that the epitope recognized by mAb J was in the linear amino acid sequence P1.The mAb J is very specific to Salmonella enterica subsp.enterica PagC and cannot react with PagC from Proteusbacillus Vulgaris,E.Coli 01,Shigella sonnei.mAb J could be used as a candidate monoclonal antibody for develop a blocking ELISA to detecte antibody of Salmonella enterica subsp.enterica.3.Development of the monoclonal antibodies against synthetic peptide P2There was no monoclonal antibody could bind to P2,so KLH-P2 were used to immune BALB/c mices.Finally,we got two monoclonal antibodties(mAbs)by cell fusion and subcloning,named mAb 1G6 and mAb 3E,indirect ELISA titers in supernatant is 1:1600 and 1:3200,respectively.Western blot analysis revealed that two mAbs were highly specific to P2,indicating that the monoclonal antibody against P2 was successfully prepared.To sum up,this study successfully got one monoclonal antibody J could bind to P1 and two monoclonal antibodies from 1G6 and 3E5 could bind to P2.It has important value in the research and diagnosis of Salmonellosis.