摘要
目的建立检测泡球蚴感染小鼠IL-17的实时荧光定量RT-PCR方法,并用于泡球蚴病小鼠IL-17基因检测。方法通过腹腔注射100μl泡球蚴组织匀浆液建立泡球蚴感染小鼠模型,感染1个月后处死,提取肝脏总RNA,反转录得到cDNA;登陆GenBank,得到IL-17基因（U43088.1）全序列,选取保守序列,应用pri mer 5设计引物,β-actin作为内参,以10倍梯度稀释IL-17 PCR产物为标准品,进行实时荧光定量RT-PCR以确定方法的特异性和灵敏度;绘制标准曲线,确定IL-17基因检测的最佳条件。结果用建立的实时定量RT-PCR检测IL-17基因无非特异性扩增,各样品熔解温度均在81.0℃,熔解峰单一;mRNA检出限为1×102pg,106~102pg总RNA均有扩增;泡球蚴感染1个月后,IL-17基因表达显著增加,感染组和对照组分别为（0.008728±0.000984）和（0.003823±0.00082）,差异有统计学意义（P〈0.05）。结论成功建立了小鼠源IL-17基因实时定量RT-PCR检测体系。经检测,小鼠感染泡球蚴1个月后IL-17基因表达增加,表明IL-17在泡球蚴感染免疫调节中可能起重要作用。
Objective To establish a method of RT-q-PCR for detection of IL-17 in mice infected with Echinococcus multilocularis.Methods To establish the mouse model,100 μ l homogenized metacestodes were injected into the anterior lobe of the liver of laboratory mice.After 1 month,the animals were sacrificed and the liver was removed to extract total RNA.cDNA was obtained by reverse transcription.A primer was designed based on the nucleotide sequence of the IL-17 gene from GENE BANK（U43088.1）by using primer 5.β-actin served as an internal control.cDNA of the liver was obtained to determine the sensitivity of RT-PCR and prepare a standard curve.Results The designed primer was specific for RT-q-PCR detection of IL-17 and the melting temperature for each sample was 81.0 ℃.The RT-q-PCR assay had a detection limit of 1×102 pg mRNA,with a dynamic range of detection from 1×106 pg to 1×102 pg mRNA.The expression of IL-17mRNA increased significantly（P0.05） in the experimental group（0.008728±0.000984） compared to the control group（0.003823±0.00082）at 1 month.Conclusion The established RT-q-PCR assay for detection of the IL-17 gene has high specificity and sensitivity.IL-17 may play an important role during E.multilocularis infection.
出处
《中国病原生物学杂志》
CSCD
2011年第1期35-38,共4页
Journal of Pathogen Biology
基金
国家自然科学基金资助项目(No.30960342)
新疆包虫病基础医学重点实验室开放课题资助项目(No.XJDX0202-2009-03)
