热敏肠毒素促进产肠毒素大肠杆菌对小肠上皮细胞系IPEC-J2的黏附的初步研究  

HEAT-LABILE ENTEROTOXIN ENHANCES ETEC ADHERENCE TO PORCINE SMALL INTESTINE EPITHELIAL CELL LINE IPEC-J2

作　　者：郁磊[1,2] 吴娟[1,3] 朱国强[1] 

机构地区：[1]扬州大学兽医学院,扬州225009 [2]南京农业大学动物医学院,南京210095 [3]南京天邦生物科技有限公司,南京211102

出　　处：《中国动物传染病学报》2012年第6期29-35,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(30571374;30771603;31072136;31270171);江苏省属高校自然科学重大基础研究项目(08KJA230002);江苏高校优势学科建设工程资助项目;教育部创新团队;科技部转基因生物新品种培育重大专项(2009ZX08006-004B)

摘　　要：本试验利用PCR技术,以K88ac标准株C83902基因组DNA为模板扩增出热敏肠毒素(heat-labile toxins,LT)基因,大小约1.1 kb。将其克隆入表达质粒载体pACYC184,构建和筛选出含正确插入LT基因的pACYC-LT重组质粒。采用同样方法,构建和筛选出两种含点突变LT基因的重组质粒(pACYC-LT72和pACYC-LT192)。进一步将上述重组质粒DNA转化入不表达任何毒素的大肠杆菌SE5000株。GM1-ELISA结果表明,上述重组菌均能在体外正常表达LT毒素蛋白。以猪小肠上皮细胞系IPEC-J2为模型,比较了表达和不表达LT的细菌对细胞黏附性能。数据表明,LT的表达使细菌对肠上皮细胞的黏附效应明显增加(12.3±3.4倍)。两种LT毒素蛋白的单氨基酸突变体的表达证明了LT毒素的ADP核糖基化作用对其增强致病菌对肠细胞的黏附作用是必要的。蛋白激酶A的抑制剂Rp-cAMP、腺苷酸环化酶的抑制剂DDA和LT毒素的受体GM1都可阻断LT毒素对细菌黏附性能的提升作用。The LT gene with size of 1.1 kb was amplified in PCR using the genomic DNA template of K88ac E.coli strain C83902.The PCR products were digested with the restriction enzymes at each end and then cloned into vector pACYC184.The recombinant LT plasmids were then constructed and confirmed by restriction endonuclease analysis.In addition,two LT variants（pACYC-LT72 and pACYC-LT192）were also constructed,each bearing mutations of amino acid residues in the A subunit（A72R and R192G） that rendered the toxin inactive.The LT expression was confirmed in GM1-ELISA.Porcine small intestine epithelial cell line IPEC-J2 with glycoprotein receptors for bacterial in vitro adhesion was prepared and used in the study.The data demonstrated that elaboration of LT promoted a significant increase in E.coli adherence and the ADP-ribosylation activity was necessary for this process.We also demonstrated that the enhancement of bacterial adherence was blocked by protein kinase A inhibitor Rp–cAMP,adenylate cyclase antagonist DDA and LT receptor GM1.

关 键 词：热敏肠毒素 黏附 IPEC-J2 

分 类 号：S852.612[农业科学—基础兽医学]