摘要
为探究旋毛虫肌幼虫中是否存在谷氨酰胺依赖性抗酸机制(AR4),本研究根据GenBank中旋毛虫肌幼虫谷氨酰胺酶(TsGLS)基因序列设计3条特异性siRNA分子,并通过浸泡法分别导入旋毛虫肌幼虫体内,利用荧光定量PCR(qPCR)、western blot以及间接免疫荧光试验(IFA),筛选沉默效果最佳的siRNA分子,并确定其最佳转染条件,结果显示,siRNA-881为基因沉默最佳的siRNA分子,且转染12 h、2μmol/L浓度、培养3 d为最佳基因沉默条件。在不同的酸性条件下培养并检测转染siRNA后旋毛虫肌幼虫的存活率,进一步将转染siRNA-881的旋毛虫肌幼虫接种小鼠,分析TsGLS基因沉默对减虫率、保姆细胞壁厚度及子代肌幼虫基因转录水平的影响。转染siRNA-881后旋毛虫肌幼虫的存活率结果显示,在pH 2.5培养2 h时的存活率明显低于其他酸性环境下;转染siRNA-881的旋毛虫肌幼虫接种小鼠后7 d旋毛虫成虫减虫率和35 d旋毛虫肌幼虫减虫率分别为61.64%和66.71%;将保姆细胞壁厚度与PBS对照组比较,siRNA-881组(15.90±1.301)保姆细胞壁厚度降低了39.78%;siRNA-881转染旋毛虫肌幼虫后,子一代肌幼虫TsGLS基因的转录水平较亲代相比降低30.30%。以上结果表明,旋毛虫具有类似于大肠杆菌中存在的谷氨酰胺依赖性抗酸系统(AR4),抑制旋毛虫TsGLS基因表达可明显降低旋毛虫肌幼虫抗酸能力。本研究首次证实通过RNA干扰技术可有效抑制旋毛虫肌幼虫TsGLS基因的表达,使该虫的抗酸能力减弱,且在宿主肠道的定植能力下降,该结果为研究旋毛虫的致病机制及其感染的防治提供新思路。
In order to explore whether there is a glutamine-dependent acid resistance mechanism(AR4) in Trichinella spiralis muscle larvae,we designed three specific siRNA molecules based on the gene sequence of TsGLs in GenBank and introduced them into the Trichinella spiralis muscle larvae by immersion method.Using fluorescence quantitative PCR(qPCR),western blot and immunofluorescence signal analysis,the siRNA molecule with the best silencing effect was selected as siRNA-881,and the optimal transfection conditions was determined.The results showed that siRNA-881 was the best siRNA molecule for gene silencing,and transfection for 12 h,2μmol/L concentration,and 3 d of culture were the best conditions for gene silencing.The survival rate of muscle larvae transfected with siRNA was determined under different acidic culture.The siRNA-881-transfected Trichinella spiralis larvae were inoculated into mice,and the effects of TsGLS gene silencing on worm reduction rate,the thickness of the nurse cytoderm and gene transcription level of offspring muscle larvae were investigated.The survival rate of muscle larvae transfected with siRNA-881 of Trichinella spiralis culturing at pH2.5 for 2 h was significantly lower than that in other acidic environments.After inoculating mice with siRNA-881-transfected Trichinella spiralis muscle larvae,the reduction rates of adult Trichinella spiralis at 7 d and Trichinella spiralis muscle larvae at 35 d were 61.64% and 66.71%,respectively.Comparing the thickness of the nurse cytoderm with the PBS control group,the thickness of the siRNA-881 group(15.90±1.301) was reduced by 39.78%.After siRNA-881 transfection in Trichinella spiralis muscle larvae,the gene transcription level of the offspring was 30.30% lower than that of the parents.The results showed that Trichinella spiralis had a glutamine-dependent acid resisting system(AR4) similar to that of E.coli,and the use of RNA interference technology could significantly reduced the acid resisting ability of Trichinella spiralis muscle larvae.This stud
作者
孟诗
张妍
杨啸
赵亚
高远
王爽
刁子洋
郭琨
宋铭忻
MENG Shi;ZHANG Yan;YANG Xiao;ZHAO Ya;GAO Yuan;WANG Shuang;DIAO Zi-yang;GUO Kun;SONG Ming-xin(Heilongjiang Key Laboratory for Zoonosis,Northeast Agricultural University,Harbin 150030,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第1期73-79,共7页
Chinese Journal of Preventive Veterinary Medicine
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(SKLVEB2018KFKT008)
国家寄生虫资源库(NPRC-2019-194-30)。
关键词
旋毛虫肌幼虫
谷氨酰胺酶
RNA干扰
抗酸
Trichinella spiralis muscle larva
glutaminase
RNA interference
acid
