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

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高旋毛虫的抗酸能力,而Ts ODC多抗血清、姜黄素和雷帕霉素则通过降低Ts ODC基因的转录和表达水平消弱旋毛虫的抗酸能力;Ts ODC可能是分泌蛋白且与旋毛虫的生长发育和繁殖有关。综上所述,本研究首次证实在旋毛虫肌幼虫中存在AR5,且其中的Ts ODC能够正调控旋毛虫的抗酸作用,为深入研究旋毛虫抗酸机制提供参考依据。

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 expression levels were decreased by 11.6%,25.8%(P<0.05)and 47.8%(P<0.01),respectively.IFA showed that the green fluorescence signal mainly appeared in the epidermal layer,the labiomastoid process of the head and the reproductive primordium of the tail in ML.These results indicated that arginine could promote acid resistance of T.trichinella by increasing the TsODC gene transcription and expression level,while TsODC polyantiserum,curcumin and rapamycin could reduce acid resistance of T.trichinella by decreasing the TsODC gene transcription and expression level.TsODC may be a secreted protein and related to the growth,development and reproduction of T.spiralis.In conclusion,this study confirmed the existence of AR5 in T.spiralis ML for the first time,and TsODC in ML can positively regulate the acid resistance of T.spiralis,providing a reference for further study of the acid resistance mechanism of T.spiralis.