表达肠出血性大肠杆菌O157 O-抗原重组减毒沙门菌的构建及其免疫效果评价  

Construction and evaluation of recombinant attenuated Salmonella expressing enterohemorrhagic Escherichia coli O157 and evaluation of its immune efficacy

作　　者：陈歆丹 张晓荟 高宇杰 王温馨 葛同鑫 宋厚辉[1] 韩月 程昌勇[1] CHEN Xin-dan;ZHANG Xiao-hui;GAO Yu-jie;WANG Wen-xin;GE Tong-xin;SONG Hou-hui;HAN Yue;CHENG Chang-yong(China-Australia Joint Laboratory for Animal Health Big Data Analytics,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,Zhejiang Engineering Research Center for Veterinary Medicine and Health Diagnostics&Advanced Technology,Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,College of Animal Science and Technology&College of Veterinary Medicine,Hangzhou 311300,China)

机构地区：[1]浙江农林大学动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地中澳动物健康大数据分析联合实验室,浙江杭州311300

出　　处：《中国预防兽医学报》2024年第12期1261-1269,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2023YFD1801000);国家自然科学基金(32473033、3210190282);浙江省自然科学基金(LQ24C010005、Q24C010018)。

摘　　要：为构建表达肠出血性大肠杆菌(EHEC)O157 O-抗原的重组减毒鼠伤寒沙门菌,本研究以EHEC O157基因组为模板,采用PCR分3段扩增O157 O-抗原基因簇(galF~gnd),并采用同源重组技术将EHEC O157 O-抗原基因簇与含asd基因的pSL2161线性化片段重组,构建表达EHEC O157 O-抗原的重组质粒pSL2161-O157并经PCR与测序鉴定正确后电转化4基因缺失沙门菌ATCC14028ΔcrpΔcyaΔasdΔrfbP,构建表达EHEC O157 O-抗原的重组减毒沙门菌O157-ATCC14028ΔcrpΔcyaΔasdΔrfbP,经PCR及测序鉴定。采用裂解法粗提野生型鼠伤寒沙门菌ATCC14028、EHEC O157与构建的重组减毒沙门菌种中的脂多糖(LPS),采用western blot鉴定上述3种菌中的LPS与EHEC O157 O-因子血清的反应性,并根据该重组菌与野生菌株ATCC14028的OD600nm值测定二者的体外生长能力。PCR与测序鉴定结果显示,重组减毒沙门菌正确构建。Western blot结果显示,重组减毒沙门菌和野生型EHEC O157的粗提LPS均能够与EHEC O157 O-因子血清反应,出现LPS特异性的梯状条带,ATCC14028株与O157 O-因子血清无反应条带。生长曲线结果显示,重组减毒沙门菌与ATCC14028株的生长速度无明显差异。表明EHEC O157 O-抗原基因簇在4基因缺失沙门菌中获得了表达,且该表达与4基因的缺失均不影响沙门菌的体外生长能力。采用首免-加强免疫策略对ICR小鼠分别口服免疫重组减毒沙门菌和4基因缺失沙门菌,二免后13 d采用间接ELISA检测各组小鼠血清中LPS的IgG抗体水平,利用荧光定量PCR(qPCR)检测各组小鼠脾细胞中细胞因子的转录水平。二免后14 d采用10 LD_(50)的EHEC O157攻毒,15 d内观察并记录各组小鼠的临床症状与死亡率,评估该重组菌株对免疫小鼠的保护效果。ELISA与q PCR结果显示,与阴性对照组和4基因缺失沙门菌免疫组相比,重组减毒沙门菌诱导小鼠产生的针对EHEC O157与ATCC14028 LPS的IgG抗体水平均极显著升高(P<0.001、P<0.01);该组小鼠To construct a recombinant attenuated Salmonella Typhimurium expressing enterohaemorrhagic Escherichia coli(EHEC)O157 O-antigen.The recombinant plasmid pSL2161-O157,which expresses EHEC O157 O-antigen,was developed by integrating EHEC O157 O-antigen cluster(galF-gnd)into the plasmid pSL2161 containing the asd gene,using homologous recombination.After verification of PCR and sequencing,the recombinant plasmid was electro-transformed into the 4-genedeletion Salmonella strain ATCC14028ΔcrpΔcyaΔasdΔrfbP,resulting in the O157 O-antigen expressing strain O157-ATCC14028ΔcrpΔcyaΔasdΔrfbP.Crude extraction of lipopolysaccharides(LPS)in wild-type Salmonella Typhimurium ATCC14028,EHEC O157 and constructed recombinant attenuated Salmonella using lysis method,the reactivity of LPS in the recombinant attenuated Salmonella to the serum against EHEC O157 O-factor was identified by western blot,and the in vitro growth capacity of the recombinant and the wild strain was determined according to the OD600nm value.The results of PCR and sequencing showed that the recombinant attenuated Salmonella was correctly constructed.The results of western blot showed that the crude LPS of recombinant attenuated Salmonella and wild-type EHEC O157 could react with EHEC O157 O-factor serum,and there was a LPS-specific ladder band,while ATCC14028 had no reaction band with O157 O-factor serum.The growth curve results showed that there was no significant difference in the growth rate between the recombinant attenuated Salmonella and ATCC14028.The results showed that EHEC O157 O-antigen gene cluster was expressed in 4 gene deletion Salmonella,and the expression and 4 gene deletion did not affect the growth capacity of Salmonella in vitro.Subsequently,female ICR mice were orally immunized with the recombinant and 4-gene-deletion Salmonella strains using a primary-booster immunization strategy,respectively.On the 13th day after the second immunization,the IgG antibody level of LPS in serum of each group was detected by indirect ELISA,and the tran

关 键 词：减毒沙门菌 疫苗递呈载体 O-抗原 肠出血性大肠杆菌O157 免疫保护 

分 类 号：S852.61[农业科学—基础兽医学]