鸡Akt基因mRNA SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立

Establishment of SYBR GreenⅠreal-time RT-PCR for detection of chicken Akt gene mRNA

为快速检测鸡细胞的增殖能力,利用SYBR GreenⅠ实时荧光定量RT-PCR技术,建立鸡Akt基因mRNA的定量分析方法。根据GenBank中原鸡Akt基因和鸡β-actin基因序列,设计合成特异性引物,应用RT-PCR技术从DF-1细胞中扩增出目的基因的DNA片段,并将纯化后的PCR产物与pMD18-T载体连接后转化大肠杆菌DH5α,构建重组质粒pMD18-Akt和pMD18-β-actin。分别以重组质粒pMD18-Akt和pMD18-β-actin为标准品建立SYBR GreenⅠ实时荧光定量RT-PCR检测的标准曲线。结果显示,建立的RT-PCR标准曲线具有良好的线性关系和特异的单个峰,能够对样品进行稳定可靠的定量检测。采用本方法检测DF-1-PrP和DF-1-count细胞系的Akt基因的相对表达量,前者为1.656 9×103,后者为2.257 4×102,DF-1-PrP细胞系的Akt基因表达量明显高于DF-1-cont细胞系(P<0.01),说明PrPC及Akt在DF-1细胞系中的表达量呈正相关。结果表明,本研究建立的鸡Akt RT-PCR检测方法能够通过对Akt基因的转录量进行检测而评价鸡细胞的增殖能力。

For rapid detection of chicken cell proliferation,based on the SYBR GreenⅠreal-time quantitative RT-PCR,aquantitative analysis method was established to analyze the quantitative of Akt mRNA in chickens.Two pairs of primers were separately designed based on sequences of Akt gene andβ-actinin GenBank.Two target DNAs were obtained by reverse transcriptase polymerase chain reaction(RT-PCR)from DF-1.Two purified target DNAs were cloned into the pMD18-T vector and transformed into Escherichia coli DH5α,respectively.The recombinant plasmids pMD18-Akt and pMD18-β-actin were acquired and served as templates to conduct the standard curves of the SYBR GreenⅠreal-time RT-PCR.In result,the two standard curves for the detection of Akt andβ-actin mRNAs were good correlation with Ct(threshold cycle)with single peak,indicating that two methods were reliable and stable.Using these methods,we detected relative expression of Akt gene between DF-1-PrP cells and DF-1-cont cells,and the expression of Akt gene in DF-1-PrP(1.656 9×103)cells was obviously higher(P<0.05)than DF-1-cont(2.257 4×102),suggesting apositive correlation between PrPcand Akt.In conclusion,the RT-PCR method can detect the transcription of Akt gene and evaluate the proliferation of chicken cells.