Prokaryotic Expression And Character Analysis Of Streptococcus Agalactiae α-Enolase

In this study, a-enolase protein has been cloned and prokaryotic expressed using Streptococcus agalactiae genome DNA which was isolated from Hainan Province. The amino acid sequence of a-enolase gene was chartered by using bio-information softwares. Further more, the enzymatic activity, plasminogen binding activity of the recombinant protein have been identified to confirm the bioactivity of it. Surface-location and adherence ability of the a-enolase protein was used to investigate the immune protection of the protein to mice. The purpose of this study is to acquire the information of the recombinant protein and to find effective vaccine component of Streptococcus agalactiae.Firstly, the cloning vector pMD19-T-a-Eno was successfully prepared. Then molecular characterization of the gene was analyzed by using bio-information sofewares. The results showed the amino acid sequence of Streptococcus agalactiae a-enolase cloned presents highly conserved among other 10 pathogenic streptococcus. Moreover, Streptococcus agalactiae a-enolase was located on the outer membrane and was hydrophobic. The plasminogen binding motif on the C terminal of a-enolase FYDAERKVY was presented on the amino acid sequence of Streptococcus agalactiae.Expression vector pET32a (+)-a-Eno was built sequentially and recombinant protein was expressed using BL21 (DE3). The results demonstrated recombinant Streptococcus agalactiae a-enolase was a protein which has a length of 67.2KDa and the optical of expression condition was at 37℃ for 3h, with 0.2mmol/L IPTG. The protein was then purified with affinity chromatography and biofuncional protein was gained after protein renaturation.The purified recombinant protein was used to test its biofunctions. Firstly, the purified protein showed enzymatic activity which could convert phosphoglycerate to phophoenolpyruvate. Besides, recombinant protein presents plasminogen binding activity. Recombinant protein was immuned to rabbits to gain anti-enolase serum and the IgG was analysed by western-blot and agar diffusion test, which proves the immunogenicity of a-enolase. The titer of the anti-serum was 1:16. The prepared anti-serum was subsequencially used to confirm the locationization of a-enolase on the Streptococcus agalactiae. Flow cytometry and extraction of the cell-wall associated protein both suggested a-enolase was located on the suface of Streptococcus agalactiae. The result of a-enolase adhere to the surface of EPC cell depicts when preincubated with anti-enolase serum, the adherence ability was decrease to a percentage of 31.89%, which indicated the contribution that a-enolase made to the adherence to EPC cell.