龙陵黄山羊羊源E.coli ClpV1基因缺失株构建及部分生物学特性分析  

Construction and partial biological characteristic analysis of Longling Yellow goat-derived E.coli ClpV1 gene deletion strain

作　　者：茶金龙 曹国春 彭洁[1] 杨维林 保志鹏[1] 胡媛媛[1] 吕念词 富国文[2] 张自芳[1] 叶妍琳 CHA Jinlong;CAO Guochun;PENG Jie;YANG Weilin;BAO Zhipeng;HU Yuanyuan;LYU Nianci;FU Guowen;ZHANG Zifang;YE Yanlin(Yunnan Vocational College of Agriculture,Kunming 650031,China;Yunnan Agricultural University,Kunming 650201,China;Longling County Animal Husbandry Workstation,Longling 678300,China)

机构地区：[1]云南农业职业技术学院,昆明650031 [2]云南农业大学,昆明650201 [3]龙陵县畜牧工作站,云南龙陵678300

出　　处：《黑龙江畜牧兽医》2024年第15期66-72,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31960692);云南农业职业技术学院项目(Ynavc202221)。

摘　　要：为了探讨龙陵黄山羊羊源E.coli ClpV1基因对E.coli部分生物学特性的影响,试验以腹泻龙陵黄山羊肛门拭子为病料进行细菌分离鉴定;从分离菌中挑选对卡那霉素和大观霉素均敏感的原菌株E.coli D1,运用CRISPR/Cas9技术构建缺失株E.coliΔClpV1-D1;连续传15代后运用PCR方法检测缺失株E.coliΔClpV1-D1遗传稳定性;挑取原菌株E.coli D1与缺失株E.coliΔClpV1-D1单克隆接种于LB液体培养基中培养,在24 h内每隔1 h测定其生长速度;30℃培养72 h后采用结晶紫染色法测定原菌株E.coli D1与缺失株E.coliΔClpV1-D1生物被膜形成能力;采用MTT法测定原菌株E.coli D1与缺失株E.coliΔClpV1-D1在第3,6,9,12小时时的细胞存活率;分别取1.0×10^(5)cfu/mL、1.0×10^(8)cfu/mL浓度的原菌株E.coli D1与缺失株E.coliΔClpV1-D1对昆明小鼠进行腹腔注射,24 h后记录各组小鼠死亡数量,进行致病性分析。结果表明:从16份肛门拭子病料中分离得到12株E.coli;运用CRISPR/Cas9技术成功构建出缺失株E.coliΔClpV1-D1;缺失株E.coliΔClpV1-D1连续传15代后仍能稳定遗传;原菌株E.coli D1和缺失株E.coliΔClpV1-D1在24 h内生长速度无显著差异(P>0.05);缺失株E.coliΔClpV1-D1的生物被膜形成能力显著低于原菌株E.coli D1(P<0.05);在测定的4个时间段缺失株E.coliΔClpV1-D1对IPEC-J2细胞毒性减弱,两种浓度的缺失株对小鼠的致死率均显著低于原菌株(P<0.05)。说明缺失ClpV1基因对E.coli生长速度无明显影响,但会降低其生物被膜形成的能力和致病性。In order to explore the effects of Longling Yellow goat-derived E.coli ClpV1 gene on some biological properties of E.coli,in the experiment,anal swabs of Longling Yellow goats with diarrhea were used as materials for bacterial isolation and identification;E.coli D1 strain(i.e.the original strain E.coli D1),which was sensitive to both kanamycin and spectacularin,was selected from the isolates,and a deletion strain of the E.coli ClpV1 gene(i.e.the deletion strain E.coliΔClpV1-D1)was constructed by using CRISPR/Cas9 technology.The genetic stability of the deletion strain E.coliΔClpV1-D1 was tested by PCR after 15 consecutive generations.The original strain E.coli D1 and the deletion strain E.coliΔClpV1-D1 were selected and inoculated in LB liquid medium,and their growth rates in the external environment were measured at 1 h intervals for 24 h.The original strain E.coli D1 and the deletion strain E.coliΔClpV1-D1 were cultured at 30℃for 72 h,and then the ability of biofilm formation was determined by crystal violet staining.Cell viability of the original strain E.coli D1 and the deletion strain E.coliΔClpV1-D1 at 3,6,9,and 12 h was determined by MTT assay.The original strain E.coli D1 and the deletion strain E.coliΔClpV1-D1 at concentrations of 1.0×10^(5)cfu/mL and 1.0×10^(8)cfu/mL were injected intraperitoneally into Kunming mice,respectively;the number of deaths in each group was recorded after 24 h,and the differences in pathogenicity were analyzed.The results showed that 12 strains of E.coli were isolated from 16 anal swabs;the deletion strain E.coliΔClpV1-D1 was successfully constructed using CRISPR/Cas9 technology;the deletion strain E.coliΔClpV1-D1 was still stable and inheritable even after 15 consecutive generations.There was no significant difference in growth rate between the original strain E.coli D1 and the deletion strain E.coliΔClpV1-D1 at 24 h(P>0.05);the ability of the deletion strain E.coliΔClpV1-D1 to form biofilm membranes was significantly reduced(P<0.05).The toxicity of the deletion

关 键 词：龙陵黄山羊 大肠杆菌 CRISPR/Cas9 ClpV1 致病性 

分 类 号：S827[农业科学—畜牧学] S852.61+2[农业科学—畜牧兽医]