2-型猪链球菌谷氨酸脱氢酶的表达和免疫学活性研究

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

目的表达2-型猪链球菌(Streptococcus suis serotype 2,SS2)的谷氨酸脱氢酶(glutamatedehydrogenase,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基因片段,插入表达载体pET-30b(+)中,构建重组表达载体pET30b-gdh。该载体经酶切鉴定和DNA测序鉴定后,转化E.coli Rosetta,IPTG诱导表达,镍离子亲和层析纯化重组蛋白。蛋白免疫印迹(western blot)证实目的蛋白的分子量和免疫反应原性。重组蛋白免疫小鼠后收集多抗血清,间接原位末端标记法(ELISA)检测其效价。结果 PCR扩增的gdh基因长度约为1300碱基对(bp),所构建重组表达载体酶切鉴定无误、测序证实碱基序列100%正确且保持正确读框。IPTG诱导表达,目的蛋白表达量约占菌体总蛋白的14.2%。亲和层析获得目的蛋白,纯度达80%以上。Western blot检测证实该蛋白能与感染SS2的患者血清发生阳性反应。重组蛋白免疫小鼠后血清效价达1∶25600以上。结论成功构建了表达载体pET30b-gdh,可在工程菌E.coli Rosetta中表达;表达蛋白具有良好的免疫学活性。

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.