Cloning And Expression Of Portective Antigen In Streptococcus Agalactiae Isolated From Bovine Mastitis

Objective: In order to handle the infection of Streptococcus agalactiae in dairy farm fromXinJiang,the study have isolated and identified in S.agalactiae from different dairy farms.Toestablish a technology platform for molecular cloning,expression,purification and immunologicalstudies of S.agalactiae,the study have cloned and expressed sip gene with genetic engineeringtechnology.Thus,the oject of this study is to explore immunogenicity of sip gene and providetechnical support for the development of subunit vaccine in S.agalactiae.Methods: In this study,The262milk samples were collected randomLy from four dairyfarms in XinJiang,then positive samples were detected by CMT.Positive samples were inoculatedon selective Granada medium to isolate S.agalactiae,and PCR and biochemical test were carriedout to indentify S.agalactiae strains.A pair of specific primer was designed based on published Sipgene sequence in the GenBank(accession number AF151361.1).Then Sip gene of S.agalactiaestrain KT639was amplified by PCR,and were inserted into plasmid pET-22b to construct arecombinant expression vector pET-22b-Sip.The recombinant plasmid was transformed into E.coliBL21(DE3) after identification by PCR,double enzyme digestion and sequencing.After inducingby IPTG,expression products were analyzed by SDS-PAGE and Western blot.Purified Siprecombinant protein with Ni-NTA and then used recombinant protein as antigen to immunemice,the ELISA method was employed to detectantibody titer.Finally,immunized mice werechallenged with S.agalactiae,picked liver then made pathological section to evaluateimmunoprotection of Sip subunit vaccine against S.agalactiae infection in mice.Results: Twenty-four S.agalactiae strains were isolated from139postive mastitis milksamples,and the prevalence of S.agalactiae is17.27%.The cultural and biochemical characteristicsof isolates were the same with the reference strain.The result of sequencing and double enzymedigestion demonstrated that nucleotide sequence and the amino acid sequence of Sip from strainwere both99%homology to the published sequence in GenBank(AF151361.1).The result of SDS-PAGE showed expressed production was a recombinant protein about51kDa,with the expectedsize consistent.The result of Western blot proved that the Sip recombinant protein could bespecifically recognized by anti-S.agalactiae antibody.Preparation subunit vaccine withrecombinant protein immunized mice,the results showed that the recombinant Sip protein canstimulate specific antibody production and antibody titer of mice reached1:256000after the thirdimmunity.The challenge test indicated that recombinant Sip protein could prevent S.agalactiaeinfection to provide immunoprotection in mice.Conclusion: This study analyzed the prevalence of Streptococcus agalactiae from bovinemastitis from four different dairy farms in Xinjiang.Constructed successfully on a prokaryoticexpression with sip gene cloned from S.agalactiae KT639.And demonstrated that Sip recombinantprotein can stimulate organism to produce immune response and protective immunity.