旋毛虫鸟氨酸脱羧酶对旋毛虫抗酸能力调控的研究  

Study on the regulation and control of ODC on acid resistance of Trichinella spiralis

作　　者：侯嘉茗 杨啸 王雪莹 薄禄琪 王爽[1] 张博涵 张鉴慧 阮入琳 宋铭忻[1] 白志坤[1] HOU Jia-ming;YANG Xiao;WANG Xue-ying;BO Lu-qi;WANG Shuang;ZHANG Bo-han;ZHANG Jian-hui;RUAN Ru-lin;SONG Ming-xin;BAI Zhi-kun(Heilongjiang Key Laboratory for Zoonosis,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学动物源性人兽共患病黑龙江省重点实验室,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2023年第3期302-309,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：家畜疫病病原生物学国家重点实验室(中国农业科学院兰州兽医研究所)开放基金课题(SKLVEB202IKFKT004);国家自然科学基金(32172883);国家寄生虫资源库(NPRC-2019-194-30)。

摘　　要：为探究旋毛虫体内是否存在鸟氨酸依赖性抗酸系统(AR5)及旋毛虫鸟氨酸脱羧酶(TsODC)对旋毛虫抗酸能力的调控作用,本实验分别采用p H1.5、p H2.5、p H4.5、p H6.6的培养液以及PBS(pH7.4)作为对照组分别培养0.5 h、1 h和2 h后,通过统计旋毛虫肌幼虫的存活率、采用荧光定量PCR(qPCR)和western blot,分别筛选旋毛虫肌幼虫体外最适酸处理条件。结果显示,在酸度为p H2.5培养2 h时,肌幼虫的存活率为51%(此时死亡数与存活数基本相等)及Ts ODC基因的转录和表达水平最高。因此,p H2.5培养2 h为旋毛虫肌幼虫体外最佳酸处理条件。于培养液中分别添加不同浓度的精氨酸、Ts ODC多抗血清、姜黄素和雷帕霉素,以PBS作为对照组分别培养12 h、24 h和48 h后,采用上述最佳酸处理条件处理后,通过计算肌幼虫存活率,采用q PCR检测Ts ODC基因的转录水平并初步筛选不同制剂的最佳培养浓度及时间,并在各最佳浓度和时间培养后,再经最佳酸处理条件处理,采用western blot检测Ts ODC蛋白的表达,分析各制剂对旋毛虫抗酸能力的调控作用;从旋毛虫感染42 d的小鼠中收集肌幼虫并采集小鼠的膈肌,分别制备冰冻切片,采用间接免疫荧光试验(IFA)检测Ts ODC蛋白在肌幼虫中的分布。存活率结果显示,与PBS对照组相比,当添加浓度为10μg/mL的精氨酸培养24 h时,肌幼虫存活率提高27%,Ts ODC基因转录水平和蛋白表达水平分别提高83.3%和68%(P<0.01);当添加浓度为1 mg/mL的Ts ODC多抗血清、20μmol/L的姜黄素和2μmol/L的雷帕霉素均培养48 h时,肌幼虫存活率分别下降13%、18%和26%,Ts ODC基因转录水平分别降低24.1%、27.1%和37.7%(P<0.01)及其蛋白表达水平分别降低11.6%、25.8%(P<0.05)和47.8%(P<0.01)。IFA结果显示,绿色荧光信号主要出现在肌幼虫的表皮层、头部的唇乳突及尾部的生殖原基。上述结果表明,精氨酸可以通过上调Ts ODC基因的转录和表达水平提In order to investigate whether ornithine dependent antacid system 5(AR5)exists in Trichinella spiralis(T.spiralis)and the regulation effect of TsODC on the antiacid ability of T.spiralis,T.spiralis were cultured at pH1.5,pH2.5,pH4.5,pH6.6 and PBS(pH7.4)as control group for 0.5 hours,1 hour and 2 hours,respectively.The survival rate of T.spiralis muscle larvae(ML),fluorescence quantitative PCR and western blot were used to screen the optimal acid treatment conditions in vitro.The results showed when ML were cultured at pH2.5 for 2 hours,the survival rate of ML was 51%(the number of ML dead was equal to the number of survival)and the transcription and expression levels of TsODC were the highest.Therefore,culturing at pH2.5 for 2 hours was the best acid treatment conditions for ML in vitro.Different concentrations of arginine,TsODC polyantiserum,curcumin and rapamycin were added into the culture medium,and PBS buffer solution was used as the control group.Subsequently,ML were cultured for 12 hours,24 hours and 48 hours,respectively.After using the above optimal acid treatment conditions,the survival rate of ML was calculated,and the transcription and protein expression of TsODC gene were detected by qPCR and western blot,respectively.ML and diaphragms were collected from mice infected with T.spiralis for 42 days.Frozen slices of ML and diaphragms were prepared,respectively and the distribution of TsODC protein in ML was detected by indirect immunofluorescence assay(IFA).The results showed that compared with PBS control group,when adding arginine at the concentration of 10μg/mL for 24 hours,the survival rate of ML was increased by 27%,and the TsODC gene transcription and protein expression levels were increased by 83.3%and 68%(P<0.01),respectively.When 1 mg/mL TsODC polyantiserum,20μmol/L curcumin and 2μmol/L rapamycin were added for 48 hours,the survival rate of ML decreased by 13%,18%and 26%,respectively,and the TsODC gene transcription levels were decreased by 24.1%,27.1%and 37.7%(P<0.01),and TsODC protein exp

关 键 词：旋毛虫 旋毛虫鸟氨酸脱羧酶 抗酸性 精氨酸 姜黄素 雷帕霉素 

分 类 号：S855.9[农业科学—临床兽医学]