目的:克隆表达2型猪链球菌N-乙酰氨基葡糖-6-磷酸脱乙酰酶(NagA)的编码基因,并测定其酶活性.方法:根据GenBank中05ZYH33基因组序列设计引物,PCR扩增NagA (SSU05_1259)基因,将其克隆到pET28a载体中,构建重组质粒pET28a:NagA,转化至大肠杆菌BL21,筛选阳性转化子进行IPTG诱导表达,产物通过SDS-PAGE与质谱鉴定;利用Ni亲和层析柱对表达产物进行纯化,获得NagA重组蛋白后测定其酶活性.结果:在大肠杆菌中高效表达了NagA基因,重组表达的NagA相对分子质量约为43×103,其酶促反应最适温度为37℃,最佳反应时间为30 min,最适反应pH值为7.5,最佳底物浓度为13 mmol/L.2型猪链球菌NagA的体外酶活为124 U/mL,酶比活为78 U/mg.结论:在原核系统中表达了NagA基因,获得的NagA蛋白具有良好的酶学活性.%Objective:To clone and prokaryotically express the gene encoding N-acetylglucosamine-6-phosphate deacetylase(NagA) of Streptococcus suis serotype 2 and to measure the enzymatic activity of the recombinant protein.Methods:The gene encoding NagA was amplified by PCR from the template S.suis 05ZYH33 genomic sequence,and was cloned into vector pET28a to obtain the recombinant plasmid pET-28a:NagA,then it was transformed into E.coli BL21.The recombinant NagA protein was induced to express by IPTG,and analyzed with SDSPAGE and LC-MS/MS,purified by Ni affifnity chromatography,followed by enzymatic activity measurement.Results:NagA gene(SSU05_1259) was expressed in E.coli with Mr about 43×103.The optimum temperature,action time,pH and the substrate concentration of the purified NagA protein were 37℃C,30 min,7.5 and 13 mmol/L,respectively.Enzymatic activity of NagA was 124 U/mL,specific activity was 78 U/mL.Conclusion:The NagA gene has been expressed in prokaryotic system,and the purified recombinant protein has the best enzymatic activity in optimum experimental condition.
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