猪链球菌2型次黄嘌呤核苷酸脱氢酶基因的克隆与鉴定  

Cloning and characterization of the gene encoding IMPDH of Streptococcus suis serotype 2

作　　者：段志涛[1] 何孔旺[1] 张雪寒[1] 倪艳秀[1] 陆承平[2] 

机构地区：[1]江苏省农科院兽医所农业部畜禽疫病诊断重点开放实验室,南京210014 [2]南京农业大学动物医学院,南京210095

出　　处：《微生物学报》2006年第5期730-733,共4页Acta Microbiologica Sinica

基　　金：国家"973项目"子课题(2006CB504403);江苏省自然科学基金重点项目(BK2006721)~~

摘　　要：根据GenBank猪链球菌2型（SS2）报道序列．对江苏分离株SS2-H部分测序，发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列，该阅读框（2738～3694）编码319个氨基酸残基，分子量为33．5kDa，与已知任何基因无同源性。通过InterPro、PHD、DNAstar分析阅读框，并定向克隆至pET-32α（＋）载体中，转化至大肠杆菌BL21，表达出分子量为48kDa的融合蛋白，蛋白免疫转印可被SS2的抗血清识别，具有免疫原性；并且含有IMP dehydrogenase结构域，催化IMP生成XMP；流式细胞仪检测该蛋白可明显影响HEp-2细胞周期。Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, S. suis serotype 2 was sequenced partly in an effort to identify important virulence factors. Two new open reading frames were found located between orf2 and mrp. One of new open reading frame（2738 - 3694） that encoded a polypeptide of 319 amino acid residues with a calculated molecular mass of 33.5kDa was identified by Western blot. GenBank database search revealed that the derived amino acid sequence shared low homology with sequences of known function from other genes. Second structure was analyzed by InterPro, PHD, DNAstar software, the deduced protein had functional domains typical of IMP dehydrogenase（IMPDH）. The PCR product of the open reading frame was transformed into E. coli BL21 and the fusion protein of 48kDa was expressed. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic. IMPDH activity staining confirmed that the protein has IMPDH function and can catalyze the rate-limiting reaction of GTP biosynthesis, the NAD-dependent reduction of IMP into XMP. Flow cytometry（FCM） revealed that the protein had apparent effect on HEp-2 cell cycle.

关 键 词：猪链球菌2型 毒力因子 IMPDH 

分 类 号：S852.611[农业科学—基础兽医学] Q78[农业科学—兽医学]