K88ac^(+)产肠毒素大肠杆菌eltAB基因缺失对其生物学特性影响的研究  

The effect of K88ac^(+)enterotoxigenic Escherichia coli eltAB deletion mutant on its biological characteristics

作　　者：段强德[1,2,3] 庞胜美 朱国强 DUAN Qiang-de;PANG Sheng-mei;ZHU Guo-qiang(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses/Joint Laboratory for International Cooperation in Agriculture and Agricultural Product Safety,Ministry of Education,Yangzhou 225009,China;Jiangsu Joint Laboratory for International Cooperation in Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,教育部农业与农产品安全国际合作联合实验室,江苏扬州225009 [3]江苏省动物重要疫病和重要人兽共患病防控技术国际合作联合实验室,江苏扬州225009

出　　处：《中国预防兽医学报》2022年第6期606-610,686,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31800121)。

摘　　要：为研究热敏肠毒素(LT)对K88ac+产肠毒素大肠杆菌(K88ac^(+)ETEC)致病能力的影响,本研究利用λ-Red同源重组系统构建了eltAB基因缺失株C83902ΔeltAB,同时将重组质粒pBR322-eltAB电转化至C83902ΔeltAB菌株中获得回补株C83902ΔeltAB/pLT。比较3株菌的生长特性,结果显示相同的条件下,野生株、缺失株和回补株的生长速度无差异;采用ELISA方法检测3株菌感染仔猪肠道上皮细胞IPEC-J2后细胞内的环磷酸腺苷(cAMP)浓度,结果显示与缺失株相比,野生株和回补株感染后细胞内的cAMP浓度均极显著上升(P<0.01)。体外对IPECJ2细胞的黏附试验结果显示,缺失株的黏附能力较野生株极显著下降(P<0.01);而回补株的黏附能力得到恢复。采用RT-qPCR检测3种菌株感染IPEC-J2后炎性因子和紧密连接蛋白基因的转录水平,结果显示缺失株感染IPEC-J2细胞后,炎性因子TNF-α和IL-8的mRNA转录水平均极显著下降(P<0.01),而3株菌感染的细胞中紧密连接蛋白编码基因Claudin-1、Occludin和ZO-1的mRNA转录水平均无变化。以上结果表明,LT毒素能够促进K88acETEC菌株对IPEC-J2细胞的黏附和诱导其炎性细胞因子的分泌,但并不能破坏肠道细胞屏障的完整性。本研究为进一步阐明K88acETEC的致病机制提供了重要的参考依据,同时为防治K88acETEC的感染提供了新的策略。In order to study the effect of heat-labile enterotoxin(LT)on the pathogenesis of K88ac^(+)enterotoxigenic E.coli(K88ac^(+)ETEC),the C83902ΔeltAB mutant was constructed byλ-Red recombinant method,and the complemented C83902ΔeltAB/pLT strain was generated by introducing the recombinant plasmid pBR322-eltAB into strain C83902ΔeltAB in this study.Comparing the growth characteristics of the three strains,the results showed that there was no difference in the growth rates of wild strain,deleted strain and complementary strain under the same conditions.The intracellular 3’,5’-cyclic AMP(cAMP)level of the IPEC-J2 cells infected with three strains was detected by ELISA.The result showed that the intracellular cAMP concentration of the IPEC-J2 cells infected with wild strain C83902 and complemented strain C83902ΔeltAB/pLT increased significantly compared with C83902ΔeltAB strain.The results of in vitro adhesion test showed that the adhesion ability of the mutant was significantly lower than that of the wild strain,while the complemented strain restored its adhesion ability.RT-qPCR was used to detect the transcription level of inflammatory cytokines and tight junction proteins in three strains infected with IPE-J2 cells.RT-qPCR result showed that deletion of eltAB gene results in reduced transcription level of TNF-αand IL-8,however,it did not alter the transcription leve of the coding genes of the Claudin-1,Occludin,and ZO-1 tight junction proteins.These results demonstrated that LT toxin was able to promote the adhesion of K88acETEC strains to piglet intestinal epithelial IPEC-J2 cells and improve inflammatory cytokines production,but it didn’t damage the integrity of intestinal barrier.This study will gain insight into the pathogenesis of K88acETEC strains and provide new strategies for prevention and control K88acETEC infection.

关 键 词：热敏肠毒素 K88ac^(+)产肠毒素大肠杆菌 黏附 致病机制 

分 类 号：S852.61[农业科学—基础兽医学]