Preparation Of SipA Polyclonal Antibody Against Salmonella 14028 And Its Expression Characteristics In Infected Mouse Tissues And Macrophages

Salmonella is a zoonotic bacterium with approximately 26,00 serotypes.Salmonella,a pathogen isolated from foodborne diseases and food poisoning cases in China,has always ranked among the top three,with the highest detection rate being Salmonella typhimurium.After infected with Salmonella in livestock and poultry,symptoms such as fever,abdominal pain,and diarrhea can occur.In severe cases,it can cause abortion in pregnant female animals and cause significant economic losses to the breeding industry.SipA is one of the virulence proteins of Salmonella 14028,secreted by T3SS-1 encoded by SPI-1;SipA regulates the rearrangement of host cytoskeleton,leading to the formation of host cell membrane folds,promoting the entry of pathogens into cells,and affecting the replication of salmonella in cells.After internalization of Salmonella,SipA binds to other proteins within the vacuoles(SCVs)containing Salmonella to promote SCV maturation and perinuclear localization.Meanwhile,SipA can significantly increase the viable count of Salmonella typhimurium in isolated intestinal cells,spleen,liver,and mesenteric lymph node tissues.SipA is continuously expressed for several hours after bacteria enter macrophages,and continues to be expressed in the cytoplasm,ultimately leading to systemic infection.This study prepared SipA polyclonal antibodies and constructed a Salmonella14028 mouse infection model.After immunohybridization with SipA polyclonal antibodies,the expression characteristics of SipA in tissues were observed;Constructing a Salmonella 14028 infected Raw264.7 cell model,SipA polyclonal antibody immunoblotting reveals the expression characteristics of SipA in the cytoplasm and nucleus of cells,which helps to further understand the pathogenic mechanism of Salmonella and provides new ideas for inhibiting Salmonella infection and treatment.According to the SipA gene sequence of Salmonella typhimurium 14028 published on Gen Bank,the primer for specific amplification of SipA gene was designed,and the SipA gene was amplified by PCR.SipA was connected to the prokaryotic expression vector p ET-28 a,and transferred to E.coli BL21 strain to construct the recombinant fusion protein p ET28 a SipA.Isopropyl group-β-The expression of p ET28 a SipA was induced by Isopropyl-beta-D-thiogalactopyranoside(IPTG).The purified SipA protein was immunized to obtain antiserum.The antiserum titer was determined by ELISA,the specificity of the antibody was analyzed by Western blot,and the SipA antibody was purified by ammonium sulfate salting out.Construct a mouse model infected with Salmonella 14028,and kill the mice after 72 hours of infection.Collect tissues from the jejunum,mesenteric lymph nodes,liver,spleen,lungs,and colon of the mice,prepare paraffin sections,and observe the secretion characteristics of SipA in vivo tissues using immunohistochemistry.The cell model of Raw264.7 infected by Salmonella 14028 was constructed.The expression characteristics of SipA in Raw264.7 cells were observed by immunohistochemistry and immunofluorescence.PCR amplified a sip A gene fragment of approximately 2,065 bp,successfully constructing a recombinant protein p ET-28a-SipA and a recombinant fusion protein SipA with a size of approximately 75 k D protein was obtaincd.The purified SipA protein was immunized with large ear white rabbits to obtain SipA antiserum;After purification with ammonium sulfate precipitation method,SipA antibody was obtained.After indirect ELISA and Western blot detection,the polyclonal antibody showed a titer of 1:512,000,indicating strong specificity;Immunohistochemical results showed that the virulence protein SipA was secreted in the jejunum,mesenteric lymph nodes,spleen,liver,lungs,and colon of infected mice,especially in tissue parts with more lymphocytes and macrophage cells.The results of cellular immunohistochemistry showed that Raw264.7 cells in the Salmonella 14028 infection group had a brown cytoplasm and a brownish blue nucleus due to the expression of SipA,while there was no color change in the control group cytoplasm,indicating that SipA was expressed in the cytoplasm and translocated to the nucleus.In order to further observe the secretion of SipA in cells,cellular immunofluorescence results showed that the virulent protein SipA was extensively secreted in the cytoplasm of Raw264.7 cells and translocated to the nucleus.