Expression And Immunogenicity Of Gapc Protein Of S.agalactiae, S.dysgalactiae, S.uberis

Streptococcus is an important pathogen that causes mastitis. 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 surface antigen GapC with the GAPDH activity as the basis for an immunogen in S.agalactiae, S.dysgalactiae, S.uberis, and to evaluate its immunogenicity and potential to protect against S.agalactiae, S.dysgalactiae, S.uberis infection.Firstly, in this study, The 13 strains isolated from the Streptococcus of mastitis were analyzed by morphology, cultural character, biochemical identification and PCR assay. Then, gapC genes of S.agalactiae strain LS0310, S.dysgalactiae strain LS0312, S.uberis strain SD0306 were amplified by PCR, and were introduced into pMD18-T vector subsequently. The recombinant clones were sequenced. The nucleotide sequence of were gapC compared and analyzed. The three recombinant plasmid were constructed by inserting gapC gene into pQE-30 vector. The three recombinant plasmid were transformed into E. coli strain XL-1-Blue, and were induced with IPTG to express recombinant TRGapC,WRGapC,RFGapC fusion proein. The three expression of target protein were detected by SDS-PAGE. The GAPDH activity assay were performed to the purified recombinant protein there after. To characterize the antigenicity of the GapC protein, 150 healthy mice were randomly devided into 15 groups, immunized with the recombinant protein, inactivation vaccine and PBS respectively. Western blot assay were performed to characterize the antigenicity of the GapC protein. The Established ELISA method was employed to detect the IgG antibody titres against GapC and bacteria in mouse serum. The microagglutination tests were done with the serum from the mice immunizd by TRGapC or WRGapC or RFGapC. On day 21 after booster immunization, All of mice in the different group containing 3 recombinant protein groups, 3 bacteria groups and 3 control groups were challenged with S.agalactiae strain LS0310, S.dysgalactiae strain LS0312, S.uberis strain SD0306. The dead rate was statistically calculated. The bacteria derived from the organ livers, spleens and kidneys were isolated and identified. The immunoprotection of the three recombinant protein was evaluated accordingly.The strains isolated from the 13 Streptococcus of mastitis are identified as Gram-Positive coccobacteria, 2 strains are S.dysgalactiae, 2 strains are S.uberis, 5 strains are S.agalactiae. The results showed that 1005bp length gapC genes of S.agalactiae strain LS0310, S.dysgalactiae strain LS0312, S.uberis strain SD0306 were successfully amplified and cloned. Sequencing results showed that the three gapC genes of the isolated strains shares 96%, 94.9%, 99.9% homology in nucleotide sequence in GenBank. The high GAPDH activities and antigenicity of the expressed WRGapC, RFGapC and TRGapC in E. coli XL-1-Blue strain were comfirmed by GAPDH activity assay and Western Blot. The ELISA results showed that the IgG antibody titres against three GapC and whole cell bacteria in mouse serums reached its peak at the third week after boost immunization. The cross reaction was observed between bacteria antigen and three GapC immunization serums. The protection rate was 80% via challenging by S.agalactiae strain, 100% via challenging by S.dysgalactiae, 90% for S.uberis challenging. But protection rate was only 40% or 50% for bacteria challenging.It can be concluded the three expressed recombinant GapC protein had high GAPDH activity and immunogenicity. The GapC protein can also protect against S.agalactiae, S.dysgalactiae, S.uberis challenge to some extent. These results suggest that the three GapC protein could be an attractive target for further Streptococcus genetic engineering vaccine.