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目的筛选多房棘球绦虫(Echinococcus multilocularis)apomucin基因(Em-apo)的qPCR最佳引物,探索该基因的表达水平。方法根据Gene DB中4种Em-apo序列,设计引物并通过荧光定量PCR(q PCR)对引物进行分析,确定每对引物的特异性、PCR扩增效率。利用筛选得到的最佳引物,分别检测经阿苯达唑(5μg/ml)和胰岛素(100 ng/ml)培养处理的多房棘球绦虫原头节(1 000个)Em-apo表达水平的变化,用稀释阿苯达唑的DMSO和胰岛素的PBS作为各自的对照。结果筛选分别得到了Em-apo-1、Em-apo-2/3、Em-apo-4和内参基因actin的特异引物各1对,qPCR熔解曲线分析结果显示,每对引物的扩增产物只出现单峰,且扩增效率均为95%~101%。qPCR分析结果显示,与DMSO对照组(1.00)相比,多房棘球绦虫原头节经阿苯达唑处理后,Emapo-1、Em-apo-2/3和Em-apo-4的表达水平均有所上升,分别为1.51±0.27、1.39±0.30和1.14±0.18,三者差异无统计学意义(P>0.05);与PBS对照组(1.00)相比,原头节经胰岛素处理后,Em-apo表达水平的变化不一,其中Em-apo-1的表达水平基本不变,Em-apo-4的表达水平有所下降,而Em-apo-2/3明显下调(0.73±0.09),但三者差异无统计学意义(P>0.05)。结论本实验筛选确定的Em-apo基因的qPCR引物特异,可用于Em-apo基因表达水平的检测。多房棘球绦虫原头节经阿苯达唑和胰岛素处理后,对Em-apo-1、Em-apo-2/3和Em-apo-4的表达水平有一定的影响。
Objective To screen qPCR primer of apomucin gene of Echinococcus multilocularis (Em-apo) and explore its expression level. Methods According to the four kinds of Em-apo sequences in Gene DB, primers were designed and the primers were analyzed by qPCR to determine the specificity of each pair of primers and the PCR amplification efficiency. Using the best primer selected, the expression level of Pro-A (1 000) Em-apo in Echinococcus multilocularis cultured by albendazole (5μg / ml) and insulin (100 ng / ml) Changes were made with PBS diluted in albendazole DMSO and insulin as the respective controls. Results One pair of specific primers were obtained for Em-apo-1, Em-apo-2/3, Em-apo-4 and actin. The qPCR melting curve analysis showed that the amplification products Appear unimodal, and the amplification efficiency is 95% ~ 101%. qPCR analysis showed that the expression of Emapo-1, Em-apo-2/3 and Em-apo-4 after treatment with albendazole in the protoscoleces of Echinococcus multilocularis compared with DMSO control group (1.00) (1.51 ± 0.27,1.39 ± 0.30 and 1.14 ± 0.18, respectively) (P> 0.05). Compared with the PBS control group (1.00), the level of insulin increased , The expression level of Em-apo-1 was not changed, but the expression level of Em-apo-4 was decreased, whereas the expression of Em-apo-2/3 was significantly decreased (0.73 ± 0.09 ), But the difference was not statistically significant (P> 0.05). Conclusion The qPCR primers of Em-apo gene identified by this experiment are specific and can be used to detect the expression of Em-apo gene. The expression of Em-apo-1, Em-apo-2/3 and Em-apo-4 in Echinococcus multilocularis progenitor cells treated with albendazole and insulin had some effects.