猪链球菌2型分泌性核酸酶A(SsnA)活性检测及其基因序列的克隆与测序分析  

Activity detection and nucleotide sequencing of Streptococcus suis DnaseA(SsnA)

作　　者：张东[1] 王楷宬[2] 汤明 黄保续[2] 范伟兴[2] 

机构地区：[1]西南大学动物科技学院,重庆4007152 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]重庆市动物卫生监督总站,重庆401147

出　　处：《中国兽医学报》2007年第6期922-926,共5页Chinese Journal of Veterinary Science

基　　金：国家"948"计划资助项目

摘　　要：对8株猪链球菌2型重庆分离株的核酸酶A全基因进行克隆测序,结果表明该基因长度为3 126 bp,与Gen-Bank发表的唯一的该基因的序列相比,核苷酸同源性高于98%,推导的氨基酸同源性高于94%。根据核酸酶A基因的测序结果建立扩增片段长度为301 bp的PCR检测方法,并用甲苯胺蓝DNA酶琼脂对猪链球菌分泌性核酸酶A的活性进行检测。检测了从病猪内脏分离的猪链球菌致病株35株,33株核酸酶A基因PCR检测阳性(阳性比例为94.3%),其中有24株分泌活性检测为阳性(阳性比例68.6%);正常猪扁桃体分离株14株,PCR检测为阳性的10株(阳性比例71.4%),其中有核酸酶A分泌活性为8株(阳性比例57.1%),检测菌株中有2型猪链球菌44株,38株扩增出核酸酶A基因的片段PCR检测阳性并且分泌活性为阳性的有32株,猪链球菌1型、7型、9型、13型、1/2型各1株,均能扩增出核酸酶A基因的片段,但只有13型猪链球菌有核酸酶A分泌活性。对猪链球菌在不同生长时期核酸酶活性的检测显示,猪链球菌在培养4、8、16、32、48 h均有核酸酶A的分泌,并且C a2+和M g2+为分泌性核酸酶A的活性必需因子。In this study,the SsnA from the 8 strains of Streptococcus suis serotype 2 Chongqing isolates was amplified by PCR using 3 pairs of primer. Nucleotide sequencing and analysis results revealed that the SsnA gene was 3 126 bp. The similarity of SsnA gene and amino acid from 8 Chongqing isolates was more than 98% and 94% ,respectively. The SsnA gene was detected by PCR with the expected size was 301 bp and the S. suis nuclease activity was detected. The results showed that 35 pathogenic Sreptococcus suis strains from diseased pigs＇viscera,32 strains detected by PCR were positive and 24 strains were the nuclease phenotypes;10 strains detected by PCR were positive and 8 strains were the nuclease phenotypes in 14 detected Streptococcus suis strains from pigs＇tonsil. Thirtyeight strains detected by PCR were positive and 32 strains were the nuclease phenotypes in 44 detected Streptococcus suis serotype 2 strains. SS1 ,SS7,SS9,SS13 and SS1/2 detected by PCR were all positive but only SS13 was nuelease phenotypes. The requirement of Ca^2＋ and Mg^2＋ for SsnA activity was determined. S. suis growth study indicated SsnA could be expressed throughout all stages.

关 键 词：猪链球菌 链球菌核酸酶A 核苷酸序列测定 活性检测 

分 类 号：S852.61[农业科学—基础兽医学] Q78[农业科学—兽医学]