干扰旋毛虫GAD基因对其抗酸能力影响的研究

Effects of GAD gene interference on the acid resistance of Trichinella spiralis

为研究旋毛虫谷氨酸脱羧酶(GAD)基因在其谷氨酸依赖型抗酸系统2(AR2)中的作用,本研究根据GenBank中登录的旋毛虫GAD基因序列,设计3条靶向该基因的siRNA(GAD-siRNA1~GAD-siRNA3)及一条阴性对照NC siRNA。将这4条siRNA以2μmol/L的剂量通过浸染法分别转染旋毛虫肌幼虫(后均简写为肌幼虫),12 h后分别采用荧光定量PCR和western blot检测各siRNA的干扰效果;采用不同剂量的最佳siRNA按照上述方法转染确定最佳干扰剂量。结果显示,2μmol/L的GAD-siRNA1(后简写为siRNA1)对GAD基因的干扰效果最好。将2μmol/L siRNA1、NC siRNA分别在不同pH值的培养液中利用浸染法分别转染肌幼虫,培养不同时间后分别计算各组存活率;同时利用荧光定量PCR方法检测GAD基因的转录水平;采用比色法建立γ-氨基丁酸(GABA)的标准曲线,通过酶活性公式和标准曲线计算酸环境培养0.5 h后各组培养液中GAD的酶活性并测定其pH值。存活率结果显示,随培养时间的增加,同一pH值培养液中各组肌幼虫的存活率均降低,其中siRNA1组肌幼虫的存活率明显低于PBS和NC siRNA组(NC组)(P<0.05),其在pH2.5培养0.5 h时的存活率最低,各组肌幼虫在pH6.6培养0.5 h时的存活率均最高。荧光定量PCR结果显示,相同p H条件下,随培养时间的延长,siRNA1组肌幼虫GAD基因的转录水平均显著低于PBS和NC组。相同培养时间内,与两个对照组相比,随着培养液p H值的减小,siRNA1组肌幼虫GAD基因的转录水平均增加,且该组肌幼虫在pH2.5时GAD基因的转录水平显著高于pH6.6时的转录水平(P<0.05)。上述结果表明,酸性环境可以诱导旋毛虫肌幼虫GAD基因转录水平的升高。酶活性测定结果显示,相同pH条件下,siRNA1组肌幼虫培养液中GAD的酶活性均比PBS组和NC组低,且该组肌幼虫在pH2.5和pH4.0时GAD的酶活性显著高于pH6.6和pH8.0(P<0.05);p H值测定结果显示,siRNA1组肌幼虫培养液pH值相比其在不同pH条件下培养前均略升高,但差异不显著。上述结果表明,干扰GAD基因后会降低旋毛虫肌幼虫GAD的酶活性,且在酸性条件下该酶活性较高。将GAD基因干扰后的肌幼虫感染小鼠,感染后第7 d迫杀小鼠收集成虫,计算旋毛虫成虫减虫率,感染后42 d采用相同方法计算肌幼虫的减虫率、繁殖力指数(RCI)及每克肌幼虫数(LPG);采集各组小鼠的膈肌,制作病理切片并经显微镜观察肌幼虫的保姆细胞。结果显示,siRNA1组成虫减虫率为31.50%,肌幼虫的减虫率为49.15%,RCI为62.51±7.32,LPG为1250.22±146.48。PBS组和NC组肌幼虫的RCI、LPG则显著高于siRNA1组(P<0.05);小鼠膈肌病理切片观察可见,siRNA1组肌幼虫子代保姆细胞壁周围有大量炎性细胞,且保姆细胞裂解,而两个对照组肌幼虫子代保姆细胞壁周围无炎性细胞。表明干扰GAD基因后肌幼虫体内的抗酸能力降低,对宿主免疫系统的抵抗力减弱。本研究首次证实了旋毛虫谷氨酸依赖型抗酸机制在其抵抗酸环境过程中发挥的作用,为旋毛虫抗酸系统的全面研究奠定了基础,并为旋毛虫病的预防提供了新思路。

In order to study the role of glutamate dependent anti-acid system 2(AR2)in the anti-acid mechanism of Trichinella spiralis(Ts),three siRNAs targeting TsGAD(GAD-siRNA1-GAD-siRNA3)and one negative control siRNA(NC siRNA)were designed according to the TsGAD gene sequence available in GenBank.The four siRNAs were transfected into Trichinella spiralis muscle larvae(ML)at a dose of 2μmol/L by immersion method.After 12 hours,qRT-PCR and western blot were used to detect the interference effects of each siRNA;The optimal concentrations of the best siRNA were determined by the above methods.Our results showed that 2μmol/L GAD-siRNA1(siRNA1)had the best interference effect on the TsGAD gene.Then,ML were transfected with2μmol/L siRNA1 and NC siRNA with different pH values respectively by immersion method,and the survival rates of each group were calculated after culturing for different hours.Meanwhile,the transcription level of TsGAD gene was detected by qRT-PCR,and the effect of acidic environment on TsGAD was analyzed.The standard curves ofγ-aminobutyric acid(GABA)were established by colorimetry,and the enzyme activity of TsGAD in each group after 0.5 hours incubation in acid environment was calculated,and the pH value was measured.The results showed that the survival rate of ML in the same pH medium decreased with the increase of culture time,and the survival rate of ML in the siRNA1 group was significantly lower than that in PBS and NC siRNA group(NC group)(P<0.05).Moreover,the survival rate was the lowest when cultured at pH2.5 for 0.5 hours,and the survival rate was the highest when cultured at pH6.6 for 0.5 hours(P<0.05).The results of qRT-PCR showed that under the same pH condition,with the extension of culture time,the transcription level of the TsGAD gene in the siRNA1 group was significantly lower than that in PBS and NC groups.Comparing with the NC and PBS groups,the transcription level of the TsGAD gene of ML in the siRNA1 group was increased as the pH values of the culture medium were decreased.The results indicated that the acidic environment could induce the transcription of the TsGAD gene in ML.The enzyme activity of TsGAD treated with siRNA1 was lower than that in the PBS group and NC group when cultured at the same pH.The enzyme activity of TsGAD at pH2.5 and pH4.0 was significantly higher than that at p H6.6 and p H8.0(P<0.05).The results showed that interference with the TsGAD gene could reduce the enzyme activity of TsGAD of ML,and the enzyme activity was higher in acidic conditions.Mice were infected with T.spiralis ML whose TsGAD gene had been silenced.After7 days,the reduction rate of T.spiralis adult worms(AW)was calculated.After 42 days,the reduction rate of ML,reproductive capacity index(RCI),and the number of larvae per gram(LPG)of ML were calculated by the same method.We also collected the diaphragms of mice and the pathological sections were made.The results showed that compared with the PBS group and NC group,the AW reduction rate of siRNA1 group was 31.50%,the RCI of ML was 62.51±7.32,and the LPG was 1250.22±146.48.The reduction rate of ML was 49.15%.The pathological section of the mice diaphragm showed that there were a large number of inflammatory cells around the nurse cell of T.spiralis ML in siRNA1 group,and the nurse cells were cleaved,but there were no inflammatory cells around the ML of the nurse cells in the two control groups.The results indicated that after TsGAD gene knockdown,the anti-acid ability of muscle larvae was decreased in vivo and the resistance to the host immune system was weakened.This study confirmed the role of glutamate dependent anti-acid mechanism of T.spiralis for the first time,which laid a foundation for the comprehensive study of antiacid system of T.spiralis and provided a new idea for the prevention of Trichinosis.