猪IGF-Ⅰ mRNA实时荧光定量PCR定量标准的构建

Construction of the standards for detecting pig IGF-Ⅰ mRNA with real-time fluorescence quantitative polymerase chain reaction

采用RT-PCR的方法利用肝组织提取的总RNA制备猪胰岛素样生长因子Ⅰ(pIGFⅠ-)cDNA目的片断,与pM D-18T vector连接成重组质粒,并转化E scherich ia coli DH 5α。联合应用PCR鉴定、α互补法、限制性酶切和序列分析法鉴定其特异性。测定纯化的重组质粒A260,确定浓度并以此制备实时荧光定量PCR(FQ-PCR)梯度浓度参考标准。结果表明:pIGFⅠ-cDNA目的片断成功制备,并获得稳定的重组质粒,保持了目的片断序列的特异性和完整性,成功构建了pIGFⅠ-实时FQ-PCR的定量参考标准。

The target fragment of plGF- Ⅰ cDNA was constructed with total RNA isolated from live tissue and amplified by RT-PCR and then was linked with pMD-18T vector to construct recombined plasmid and to transformate to Escherichia coli DH5a. Its specificity was tested by direct PCR, a complementation, restriction enzyme cleavage, and sequencing method. The concentration of purified recombine plasmid by detecting absorbance in AzG0nm and then the plasmid was diluted to serial standard concentrations for FQ-PCR. The results showed that the target fragment of pIGF- Ⅰ cDNA was constructed successfully; the recombined plasmid contained the target fragment was stable and kept its specificity and sequence completion; reference standards for real-time FQ-PCR were constructed successfully.