禽致病性大肠杆菌luxS和rbsC双基因缺失株的构建及其生物特性分析  

Construction and Biological Characteristics Analysis of LuxS and RbsC in Avian Pathogenic Escherichia Coli

作　　者：涂春田 左佳坤 蒋蔚[2] 陈兆国[2] 米荣升[2] 黄燕[2] 韩先干[2] 汪洋[1] TU Chuntian;ZUO Jiakun;JIANG Wei;CHEN Zhaoguo;MI Rongsheng;HUANG Yan;HAN Xiangan;WANG Yang(College of Animal Science and Technology,Henan University of Science and Technology,L uoyang 471003,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]河南科技大学动物科技学院,洛阳471003 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2021年第1期9-15,共7页Chinese Journal of Animal Infectious Diseases

基　　金：上海市科学技术委员会科研计划项目(17140900400,18140900700);中央级公益性科研院所基本科研业务费(2019JB01);国家自然科学基金项目(31872843,31572546,31772707)。

摘　　要：禽致病性大肠杆菌(APEC)能引起家禽严重的呼吸系统和全身性疾病,由于APEC耐药菌株的不断出现迫使新型抗菌方式的研发迫在眉睫。本研究旨在评价APEC的luxS和rbsC双缺失的突变株作为减毒疫苗候选株的潜力,通过在APEC分离株DE17的单基因缺失株DE17ΔluxS的基础上,利用Red重组系统构建双基因缺失株DE17ΔluxSΔrbsC,并对其生物学特性进行了分析。结果显示:rbsC的缺失不影响DE17ΔluxS的生长特性和生物被膜形成能力,但运动性显著降低;与DE17ΔluxS相比,DE17ΔluxSΔrbsC的黏附能力和入侵能力分别降至46.92%和28.78%;半数致死量(LD50)结果显示,毒力下降了115.5倍;肝脏、脾脏和血液中的载菌量分别下降了3.63倍、79.20倍和2124.41倍。根据以上结果推测,rbsC基因的缺失会进一步降低DE17ΔluxS的毒力,本文为评价DE17ΔluxSΔrbsC株减毒机制提供参考。Avian pathogenic Escherichia coli(APEC)causes severe respiratory and systemic diseases in poultry.Due to the continuous emergence of APEC resistant strains,the development of new antibacterial methods is impressive.This study was to evaluate the DE17ΔluxSΔrbsC double-mutant strain as a vaccine candidate.Based on the DE17ΔluxS mutant strains of APEC DE17,the DE17ΔluxSΔrbsC mutant strain was constructed using a modified lambda Red recombinase method.The differences in biological characteristics such as growth curves,motilities,biofilm formation of DE17ΔluxSΔrbsC strains were compared to the strain DE17ΔluxS.The results showed that the deletion of rbsC neither affected the growth curves nor biofilm formation of DE17ΔluxS while the bacterial motility reduced significantly.Compared with DE17ΔluxS,the adherence and invasion abilities of DE17ΔluxSΔrbsC decreased to 46.92%and 28.78%,respectively.The results of 50%lethal dose showed that the virulence of DE17ΔluxSΔrbsC was further attenuated by 115.5-fold.Compared to the strain DE17ΔluxS,the bacterial loads of DE17ΔluxSΔrbsC were reduced by 3.63-,79.20-and 2124.41-fold in the livers,spleens and blood of infected chickens,respectively.These results suggested that the deletion of rbsC gene further significantly reduced the virulence of DE17ΔluxS.This study provided a basis for evaluating its attenuation mechanism.

关 键 词：禽致病性大肠杆菌 rbsC基因 RED重组 半数致死量 

分 类 号：S852.612[农业科学—基础兽医学]