Expression And Immunogenicity Of Streptococcus Uberis GapC Protein

Streptococci is an important pathogen that causes a varitety of diseases among human and animals.Due to the lack of common protective antigens among the Streptococci isolates,existing vaccines available remain ineffective in clinical trials.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) protein can be used as antigens in vaccines to protect against parasitic and microbial infections.Thus,the object of this study is to use conserved Streptococcus uberis(S.uberis) surface antigen GapC,which with the GAPDH activity,as the basis for an immunogen,and to evaluate its immunogenicity and potential to protect against Streptococcus agalactiae(S.agalactiae) and Streptococcus dysgalactiae(S. dysgalactiae) infection.Firstly, gapC gene of S.uberis was amplified by PCR,and was introduced into pGEM-T vector subsequently. pET-28a(+)-gapC recombinant plasmid was constructed by inserting gapC gene into pET-28a(+) vector. The recombinant clone, pET-28a(+)-gapC,was sequenced.The nucleotide sequence of gapC was compared and analyzed with the one published previously in GenBank.The recombinant plasmid was transformed into E.coli strain BL21(DE3),and was induced with IPTG to express recombinant GapC fusion proein.The expression of target protein was detected by SDS-PAGE.The Western blot assay were performed to the purified recombinant protein there after.To characterize the antigenicity of the GapC protein,80 healthy Kunming mice were randomly devided into 4 groups,immunized with the recombinant protein and placebo(PBS).Enzyme-Linked Immunosorbnent Assay(ELISA) was used to measure the levels of IgG antibody titres in mouse serums.Finally,the immunized mice were challenged with the S. agalactiae and S. dysgalactiae,clinical symptoms and pathological changes were observed to characterize the morbility of the challenged mice,which was then comfirmed by the isolation and identification of the S.agalactiae and S. dysgalactiae from livers,spleens and kidneys organs, respectively.The immunoprotection of the recombinant GapC proein was evaluated accordingly.The results of the study showed that 1,011bp lengthed gapC gene of S.uberis strain was successfully amplified and cloned.Sequence analysis showed that the gapC gene was shared 99.8%,91.4% and 88.9% nucleotide homology with S.uberis gapC gene sequence(accession No.AF421900), S.dysgalactiae gapC gene sequence (accession No.AF421899) and S.agalactiae gapC gene sequence (accession No.AF375662) in GENBANK. SDS-PAGE result showed that the recombinant GapC was expressed successfully in E.coli BL21(DE3) strain,the antigenicity were comfirmed by Western Blot.The IgG antibody titre against GapC in mice serums reached its peak at day 14 after boostimmunization(1:64000).10 days after the third immunization,these mice were challenged with the S.agalactiae (5×10~8CFU/200μL)and S.dysgalactiae(2.5×10~8CFU/200μL ).At the 21th day after challenge,the mice immunized could be protected from the S.agalactiae attack at rate of 100% and the S.dysgalactiae attack at rate of 95% respectively,compared with the control group.The results indicated that the recombinant protein may be applied into clinical prevention for Streptococci infections.