摘要
为研究禽致病性大肠杆菌(APEC)六型分泌系统(typeⅥsecretion system,T6SS)clpV基因对Ⅰ型菌毛表达的影响,本研究利用Red同源重组系统构建了禽致病性大肠杆菌clpV基因缺失株TW-XM△clpV。将clpV基因克隆到表达载体pBR322中,构建相应回补株TW-XMC△clpV。通过PCR特异性检测和基因测序,验证上述突变株均构建成功,且能稳定遗传。利用荧光定量PCR检测野生株、clpV缺失株和回补株中Ⅰ型菌毛9个主要基因的mRNA相对表达量。结果显示:与野生株相比,缺失株中fimC、fimF、fimG、fimI的mRNA相对表达量显著下降,fimB、fimD、fimE极显著下降,fimA与fimH则无统计学意义;而回补株各基因的mRNA相对表达量均得到了恢复。本研究表明clpV基因缺失影响了Ⅰ型菌毛的表达。本研究有助于了解T6SS与Ⅰ型菌毛合成的关系,为进一步研究APEC的致病机理奠定基础。
In order to investigate the relationship between the clpV gene of the typeⅥsecretion system(T6SS)of avian pathogenic Escherichia coli(APEC)and the expression of typeⅠfimbriae,the clpV deleted strain was constructed by the Red recombination system,the APEC TW-XM strain was used as the prototype strain.In addition,the clpV gene was cloned into pBR322to construct the complementary strain TW-XMC△clpV.The mutant strain and complementary strain were confirmed by PCR and DNA sequencing,and they proved to be genetically stable.The expression of nine typeⅠfimbriae related genes was detected by qRT-PCR.Results showed that,compared with wild type strain,the expression of fimC,fimF,fimGand fimI decreased significantly,the expression of fimB,fimD,and fimE decreased significantly,the expression of fimA and fimH had no significant difference.The expression of each gene in the complementary strain restored.This work proved that the deletion of the clpV gene had an impact on the expression of typeⅠfimbriae,it provides the basis for studying the interaction between T6SS and typeⅠfimbriae,it is also helpful in understanding the pathogenesis of APEC.
作者
钟昊然
王培莉
陈艳飞
孟霞
朱国强
李建基
崔璐莹
董俊升
王亨
ZHONG Haoran;WANG Peili;CHEN Yanfei;MENG Xia;ZHU Guoqiang;LI Jianji;CUI Luying;DONG Junsheng;WANG Heng(Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009,China;Joint International Research Laboratory of Agriculture and Agri-Product Safety,Ministry of Education of China,Yangzhou,Jiangsu 225009,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第6期1105-1110,共6页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2017YFD0500203)
国家自然科学基金资助项目(31972651)
扬州大学研究生科研创新计划资助项目(XKYCX19_138)
江苏高校优势学科建设工程资助项目(PAPD)
江苏高校品牌专业建设工程资助项目(TAPP)。
