2-型猪链球菌谷氨酸脱氢酶的表达和免疫学活性研究

目的 表达2-型猪链球菌(Streptococcus suis serotype 2,SS2)的谷氨酸脱氢酶(glutamate dehydrogenase,GDH),研究其免疫学活性,探讨其作为疫苗候选分子的可能性.方法 用聚合酶链反应(PCR)技术从SS2临床分离株05ZYH33的基因组DNA中扩增出gdh基因片...

xpression and immunologic activity analysis of glutamate dehydrogenase of S.suis 2

Objective To express glutamate dehydrogenase（GDH） of S.suis 2 and analyze its immunologic activity for exploring probability as protective antigen candidate.Methods The gdh gene was amplified from S.suis 2 clinical isolate（strain 05ZYH33） genome DNA by PCR.The gene fragment was inserted into the expression vector pET-30b（＋） to build pET30b-gdh.The recombinant vector pET30b-gdh was identified by restriction enzyme cutting and DNA sequencing,and then transformed to E.coli Rosetta for expression under IPTG induction.The obtained fusion protein was purified by Ni-NTA affinity chromatography.The immunologic activity of the purified protein was proved by immunizing mice and Western blot,respectively.Results The PCR product was about 1300 bp.The gene fragment inserted into the recombinant vector was proven to be completely identical with the sequence of the gdh gene in the whole genome sequence of S.suis 2.The target protein was expressed up to 14.2% of total somatic protein under IPTG induction.The protein purity reached more than 80% after purification.The recombinant protein could be recognized by human serum infected with S.suis 2.Antibody titer of mouse serum immunized with recombinant protein reached more than 1∶25600.Conclusion The expression vector pET30b-gdh is successfully constructed.The target protein can be overexpressed in E.coli and possess immunologic activity.