Expression And Function Of GapC Gene Of Streptococcus Uberis Isolated From Southern Xinjiang

Pathogenic microorganisms is the main factor to cause dairy cows mastitis. Streptococcus uberis asone of the main pathogens causing the disease is associated with a variety of virulence factors. Wherein thegapC gene, encoding the GapC protein, which have glyceraldehyde-3-phosphate dehydrogenase (GAPDH)activity, is considered as a virulence-associated immunomodulatory protein. GapC protein can serve as agood vaccine target sites because it plays an important role in the development of the disease and the signaltransmission. Therefore, to study the function of GapC protein of streptococcus uberis would supply a basisfor antigen screening, vaccine development and provides a theoretical guidance for mastitis in dairy cow’sdisease prevention and control.In this study, S.uberis field strain from southern Xinjiang dairy herds harm to the healthy developmentof the dairy industry as the research object.Firstly, the gapC gene of S.uberis was amplified by PCR and was cloned into the prokaryoticexpression vector pET32a (+), to construct prokaryotic expression vector pET32a (+)-gapC. The resultsshowed that0.5mmol/L IPTG induced for6h can get a lot of recombinant proteins, which are mainly ininclusion bodies.The molecular weight of Recombinant protein is about54.0KD, which was expected.Secondly, The GapC recombinant Protein Was purified by nickel ion affinity chromatography underdenaturing conditions. Emulsified with immunologic adjuvant, Preparing for vaccine and immune mice.Western-blotting detect the specific antibody binding activity, Indirect ELISA assay test the antibody titer.The mice were poison attack after immunization. The results showed that antibody titers by ELISA reached1:128000, indicating that GapC protein has good immunogenicity. By Western blot analysis,54.0KD-specific binding of the antibody to the protein, indicating that GapC protein has good reactogenicity.Challenge test results show that a high rate of protection by GapC protein immunohistochemistry, itreached70%. While, the whole-cell protection rate is not high, rate of30%.Finally, by recombinant DNA technology, build gapC gene deletion vector pFW5-gapC, therecombinant plasmid was transplanted into S.ubreis to get S.ubreis gapC gene mutants. We have learned anew electrotransformation method for the construction of the over-expression vector and induce GapCprotein over-expressed in the S.uberis. This is the basis for studay the function of GapC.In summary, GapC protein has high immunogenicity and reactogenicity, indicate that gapC gene canbe used as a candidate gene for preparation vaccine for S.uberis infection, which will provide an effectivetheoretical guidance for preventing and controlling of the cow mastitis.