摘要
旨在探究旋毛虫肌幼虫体内是否存在鸟氨酸依赖型抗酸系统,本研究根据GenBank中Ts-ODC全序列基因组设计三条特异性siRNA分子,采用脂质体浸染转染的方法将其分别转染至旋毛虫肌幼虫体内,使用qPCR、Western blot和间接免疫荧光试验,筛选最佳干扰siRNA分子及最佳干扰条件;并在pH 1.5、2.5、4.5和6.6的条件下将肌幼虫分别培养0.5、1和2 h,从肌幼虫Ts-ODC基因转录水平、酶活性及存活率等方面分析Ts-ODC基因干扰对旋毛虫肌幼虫抗酸能力的影响;将Ts-ODC基因被干扰的旋毛虫肌幼虫感染小鼠,在7和42 d时分别收集成虫和肌幼虫,分别计算其减虫率;将子一代肌幼虫在pH 2.5的酸性条件下培养2 h,计算其存活率,对Ts-ODC基因干扰的遗传性进行分析。结果显示,Ts-ODC siRNA-757为最佳干扰分子,转染12 h、浓度为2μmol·L^(-1)培养3 d时为最佳干扰条件;各组pH 2.5培养2 h时Ts-ODC基因转录水平最高,pH 1.5培养2 h时虫体的存活率明显低于其他培养条件(P<0.01),pH 4.5培养0.5 h时虫体体内酶活性最高;转染Ts-ODC siRNA-757的肌幼虫接种小鼠后,7 d时成虫和42 d时肌幼虫的减虫率分别为58.95%和65.91%;其子一代肌幼虫在pH 2.5的培养液内培养2 h时其存活率为49%。综上表明,旋毛虫肌幼虫体内具有鸟氨酸依赖型抗酸系统,本研究首次通过siRNA技术干扰旋毛虫肌幼虫Ts-ODC基因,且证实干扰Ts-ODC基因可降低肌幼虫抗酸能力,为后期旋毛虫病的防治提供新思路。
In order to explore the existence of ornithine-dependent anti-acid system in Trichinella spiralis(T.spiralis,Ts)muscle larvae,three specific siRNAs molecules were designed according to the complete genome sequence of Ts-ODC(Trichinella spiralis ornithine decarboxylase)in GenBank.Three siRNAs were transfected into T.spiralis muscle larvae by liposome transfection.The best interference siRNA molecules and the best interference conditions were determined by qPCR,Western blot and indirect immunofluorescence assays.The muscle larvae were cultured at pH 1.5,2.5,4.5 and 6.6 for 0.5,1 and 2 h,respectively.The effects of Ts-ODC gene interference on the acid resistance of muscle larvae were evaluated from three aspects of the ODC transcription level,ornithine decarboxylase activity and larvae survival rate.Mice were infected with muscle larvae of T.spiralis after Ts-ODC gene interference.Adults and muscle larvae were collected at 7 d and 42 d,respectively,and the worm reduction rates were calculated.The first-generation muscle larvae were cultured under acidic conditions at pH 2.5 for 2 h,and the survival rate was calculated to analyze the heritability of Ts-ODC gene interference.The results showed that Ts-ODC siRNA-757 was the optimal interference molecule,and the best interference conditions were transfection for 12 h and 3 d culture at a concentration of 2μmol·L^(-1);the highest transcription level of Ts-ODC gene was reached at pH 2.5 for 2 h,and the survival rate was significantly lower than other culture conditions at pH 1.5 for 2 h(P<0.01).The ornithine decarboxylase activity was the highest at pH 4.5 for 0.5 h.After the mice were orally gavaged with muscle larvae transfected with Ts-ODC siRNA-757,the worm reduction rates were 58.95%and 65.91%at 7 d and 42 d,respectively.The survival rate of the first-generation muscle larvae was 49%when cultured in a pH 2.5 medium for 2 h.The above experiments concluded that T.spiralis muscle larvae has an ornithine-dependent acid-resistant system.In this study,the Ts-ODC gene of T.spiralis muscle larvae was interfered by siRNA technology for the first time,and it was confirmed that interfering with the Ts-ODC gene could reduce the acid resistance of muscle larvae,which provided a new idea for the prevention and treatment of Trichinellosis.
作者
薄禄琪
王雪莹
侯嘉茗
张力
张鉴慧
阮入琳
张博涵
王爽
宋铭忻
张子群
BO Luqi;WANG Xueying;HOU Jiaming;ZHANG Li;ZHANG Jianhui;RUAN Rulin;ZHANG Bohan;WANG Shuang;SONG Mingxin;ZHANG Ziqun(Heilongjiang Key Laboratory for Zoonosis,Northeast Agricultural University,Harbin 150030,China;Technology Center of Harbin Customs,Harbin 150008,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2023年第5期2114-2125,共12页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(32172883)
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)开放课题(SKLVEB2021KFKT004)
国家寄生虫资源库(NPRC-2019-194-30)。
关键词
旋毛虫
鸟氨酸脱羧酶
RNA干扰
抗酸系统
Trichinella spiralis
ornithine decarboxylase
RNA interference
anti-acid system
