肠炎沙门菌glpK基因缺失株的生物学特性及其免疫效果评价的研究  

Research on the biological characteristics and evaluation of immune efficacy of glpK mutant of Salmonella enteritis

作　　者：冀梦瑶 周诗淼 蒋卉[2] 张莹辉[2] 彭小薇[2] 李佩国[1] 吴同垒[1] JI Meng-yao;ZHOU Shi-miao;JIANG Hui;ZHANG Ying-hui;PENG Xiao-wei;LI Pei-guo;WU Tong-lei(Hebei Normal University of Science&Technology,Qinhuangdao 066004,China;China Institute of Veterinary Drug Supervision,Beijing 100080,China)

机构地区：[1]河北省预防兽医学重点试验室,河北科技师范学院,河北秦皇岛066004 [2]中国兽医药品监察所,北京100080

出　　处：《中国预防兽医学报》2022年第5期471-476,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金(31902310);河北省重点研发计划(19226629D);秦皇岛市科学技术研究与发展计划(201803B006)。

摘　　要：为分析甘油激酶(GlpK)对肠炎沙门菌毒力的影响,本研究使用野生菌株c50336和glpK基因缺失菌株c50336ΔglpK进行体外模拟应激试验,分析glpK基因缺失对沙门菌抵抗应激环境的影响。将这两株菌分别以MOI 100感染鼠源巨噬细胞Raw264.7,在不同时间点计算胞内存活的细菌数,以评价glpK基因的缺失对沙门菌胞内存活率的影响;将菌株c50336和c50336ΔglpK腹腔注射小鼠,利用寇氏法计算各菌株对小鼠的半数致死量(LD;)。结果显示,与c50336相比,c50336ΔglpK在氧化和高渗环境中的存活率极显著降低(0.001<P<0.01),但在酸性和碱性环境中的存活率无明显变化,且在巨噬细胞内的存活率明显降低;LD_(50)测定结果显示,c50336ΔglpK和c50336对小鼠的LD_(50)分别为1.6×10^(7) cfu和6.7×10^(2) cfu。将菌株c50336ΔglpK以2.1×10^(6)cfu/只的剂量免疫BALB/c小鼠,14d后二免,首免28d时,以1.5×10^(5) cfu/只注射菌株c50336,计算15 d内小鼠的免疫保护率;按上述方法免疫小鼠一次,分别在免疫后3d、10d和21d,对各组小鼠采血分离血清,经间接ELISA检测小鼠血清抗体水平;取脾脏称重,计算脾脏指数;采用细菌计数法测定小鼠脾脏载菌量;制备免疫小鼠脾淋巴细胞悬液,分别经c50336ΔglpK全菌蛋白和ConA刺激,72 h后采用MTT法检测各组小鼠脾细胞的增殖能力。免疫保护率结果显示,glpK缺失株可对免疫小鼠提供100%的保护率,而攻菌对照组小鼠在攻菌后12 d全部死亡。脾脏指数和载菌量检测结果显示,与对照组相比,免疫后3d、10 d和21 d的免疫组小鼠脾脏指数均极显著升高(0.001<P<0.01),且10 d时脾脏指数最高;小鼠脾脏载菌量逐渐降低,并在免疫后21 d脾脏细菌被完全清除。血清抗体检测结果显示,免疫组小鼠抗体水平随免疫次数的增加而升高,而对照组小鼠血清抗体一直为阴性。淋巴细胞增殖能力检测结果显示,c50336ΔglpK全菌蛋白刺激的小鼠脾细胞增殖指数随免�To investigate the effect of Glycerol kinase(GlpK)on virulence of Salmonella enteritidis,the wild strain c50336 and glpK gene deletion strain c50336ΔglpK were used in simulated stress test in vitro to evaluate the influence of the gene deletion on Salmonella resistance to stress environment.Mouse-derived macrophage Raw264.7 was infected by the two strains with MOI 100respectively,and the number of viable bacteria was calculated at different time to evaluate the effect of the gene deletion on the intracellular survival rate of Salmonella.The two strains were injected intraperitoneally into mice,respectively,and half lethal dose(LD_(50))was measured using the Karber method.The results showed that,compared with strain c50336,the survival rate of c50336ΔglpK was significantly decreased in oxidized and hypertonic environments(0.001<P<0.01),but there was no significant change in acidic and alkaline environments,and the survival rate in macrophages decreased significantly.The results showed that LD_(50) value of c50336ΔglpK and c50336 was 1.6×10^(7)cfii and 6.7×10^(2)cfu,respectively.Mice were immunized with c50336ΔglpK AT at dose of 2.1 x 10^(6)cfu/mouse,and boosted after 14 days.On 28 days post first immunization,the mice were challenged with c50336 at dose of 1.5 x 10^(5)cfu/mouse.The immune protection rate was calculated within 15 days.After immunized once using the above method,blood of the mice in each group were collected at 3,10 and 21 days after immunization,respectively,for serum separation,and the serum antibody levels of mice were detected by indirect ELISA.Spleen was weighed and spleen index was calculated.Bacterial count method was used to determine the bacterial load in spleen of mice.The spleen lymphocyte suspension of immunized mice was prepared and stimulated by total bacterial protein of c50336and ConA,respectively.After 72 hours,the proliferation ability of splenocytes in each group was detected by MTT assay.The results of immune protection rate showed that c50336ΔglpK could provide 100%protec

关 键 词：甘油激酶GlpK 基因缺失株 疫苗 

分 类 号：S852.61[农业科学—基础兽医学]