Expression And Immunogenicity Research Of Fusion Protein Of S.dysgalactiae GapC And S.typhimurium FliC

The cow mastitis (Dairy Cows Mastitis) is caused by a variety of pathogenic microorganisms, andserious damage to the development of milk cow industry. Streptococcus is one of the important pathogencause of dairy cows mastitis. With the treatment of antibiotics in general use, there appear a large numberof drug-resistant strains, cause appear drug residues and treatment difficulty. So, the vaccine immunizationbecome the key and the best choice of the prevention of the mastitis. Research shows that, Streptococcusdysgalactiae GapC protein can better prevention the mice infected by the Streptococcus dysgalactiae、Streptococcus uberis and Streptococcus agalactiae. Innate immune is not only the first barrier againstpathogens invasion, but also identify and distinguish between pathogens, plays a key role in acquiredimmune. Salmonella typhimurium flagellum protein is one kind of the pathogen associated molecular pattern,having the activity of adjuvants, can be antigen presenting cell’s pattern recognition receptor, PRR to identifyand distinguish. The main function of the flagellum protein is action on Toll like receptor5cells, through theTLR5activation, activate MyD88signalling pathways, thus regulation TNF-α, IL-6and IL-12promoteinflammation cell factors synthesis. At the same time raising up the molecular collaborative stimulationCD80, CD86and Ⅱ MHC molecules, promoting the cell antigen presenting cell to active and mature, andthen activation and differentiation helper T cell, start-up acquired immune response.The proposed of this study is use E. coli expression system to express having biological functions FliCand FliC-GapC protein, construct one vaccine that can induce natural immune, at the same time can beadjusted acquired immune. Through the research and analysis of the difference between GapC and FliC-GapCthat invoke the natural immune response and acquired immune response laws, as well as to the mice immuneprotection, so as to demonstrate the flagellum protein’s adjuvant effect, at the same time providing newknowledge and new ways for the cows mastitis new vaccines.First, amplifed S.typhimurium8014strain fliC gene by PCR method, cloning to pMD18-T vector. Aftersequencing fliC gene and contrast sequence analysis, cloned fliC gene to pQE-30express vector andtranslated into E. coli XL1-Blue strain. After IPTG induction, identified the expression about fliC protein bySDS-PAGE and determine its biological activities. Secondly, through the overlap polymerase chain reaction,amplified fliC-gapC fusion gene, cloned to pQE-30express vector and translating into E. coli XL1-Bluestrain, After IPTG induction, purified fliC-gapC fusion protein by nickel column, and detected the GAPDHactivity of fusion protein. Finally, by the use of mice as experimental animal, designed the experimentalgroup and control group, through the test of antibody in serum and mice immune cell factors IL-2, IL-4,IL-10and the content of the secretion of INF-γ and lymphocyte proliferation experiment, finally attacked byS.dysgalactiae LS0312strains, evaluated fusion protein FliC-GapC immune protection effect and theflagellum protein adjuvant fuction.This research has successfully constructed biologically active FliC and FliC-GapC protein. MiceExperiment showed that the group of FliC-GapC protein, GapC protein+freund’s adjuvant immune after twoweeks the antibody titer significant difference between the control group (P <0.05); And GapC protein, FliCprotein immune group and PBS group, antibody growth is not obvious.21days after the second immunization cytokines CKs detection of the serum, INF-γ spots number and lymphocyte proliferationexperimental results consistent with the antibody titer changing regularity. Rate of immune protection againststrains attack about GapC+freund’s adjuvant, FliC-GapC, GapC, FliC, and PBS control group were80%,70%,10%,0%,0%.To sum up, prokaryotic expression FliC-GapC fusion protein can induce mice producing the specificityantibody of the GapC protein, with good immunogenicity, and the flagellum of the fusion protein play a goodimmune adjuvants fuction. The fusion protein can effectively prevent Streptococcus dysgalactiae infection, atthe same time providing reference for the cows mastitis research.