猪链球菌2型c-di-AMP水解蛋白基因gdpP的表达与活性分析  

Expression of the c-di-AMP Phosphodiesterase gdpP Gene of Streptococcus suis Serotype 2 and Its Activity Analysis

作　　者：杜斌[1] 孙建和[1] 

机构地区：[1]上海交通大学农业与生物学院上海市兽医生物技术重点实验室,上海200240

出　　处：《上海交通大学学报（农业科学版）》2015年第5期34-39,共6页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家自然科学基金项目(31172381和31372500);上海市科技兴农中重点攻关项目(2014-3-1)

摘　　要：为研究猪链球菌2型(Streptococcus suis serotype 2,SS2)环二腺苷酸(c-di-AMP)水解蛋白GdpP的生化特性,根据已公布的SS2中国强致病株98HAH33全基因组中的gdpP序列信息设计引物,以SS2强致病株HA9801基因组为模板,通过PCR方法克隆获得了SS2的c-di-AMP水解蛋白基因gdpP完整序列,其阅读框由1 965个核苷酸组成,编码654个氨基酸。应用大肠杆菌重组表达系统,表达、纯化了GdpP,进而分析了其体外生物活性,结果显示纯化的重组蛋白可以在体外水解c-di-AMP形成pApA。其水解酶活性在碱性环境条件下(pH 8.0)相对较强,而在酸性环境下(pH 6.0,6.5)未检测到活性;同时,水解酶活性还依赖于二价金属离子Mg2+、Mn2+以及Co2+的存在,且在Mn2+存在的条件下相对活性最强,而在Ca2+、Fe2+和Cu2+存在的条件下则均未表现出活性。上述结果为进一步深入研究c-di-AMP在猪链球菌2型中发挥的生物学功能奠定了基础。The gdpPgene of Streptococcus suis serotype 2（SS2）was cloned with polymerase chain reaction（PCR）and expressed in pEASY-E1/E.coli BL21（DE3）system.Affinity purified GdpP was used to evaluate its biochemical property in vitro.Results showed that,the open reading frame of gdpP was 1 965 bp,which encoded a protein of 654 amino acids.The purified recombinant proteins could hydrolyzed the cdi-AMP to pApAin vitro.The recombinant GdpP exhibited a higher enzymatic activity in the alkaline condition with pH at 8.0,while no phosphodiesterase activity was detected in the acidic conditions（pH 6.0,6.5）.In addition,the phosphodiesterase activity is dependent on divalent metal ions such as Mn2＋,Mg2＋,and Co2＋ions,and it is far greater in the presence of Mn2＋than other divalent metal,and no phosphodiesterase activity was observed in the presence of Ca2＋、Fe2＋and Cu2＋.In conclusion,theverification of the phosphodiesterase activity of the purified recombinant proteins in vitro established the foundation for further study of the biofunction of c-di-AMP in SS2.

关 键 词：环二腺苷酸 GdpP 水解酶活性 猪链球菌2型 

分 类 号：S852.61[农业科学—基础兽医学]