【摘要】 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。更多还原

【Abstract】 The target fragment of pIGF-ⅠcDNA 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 DH5α.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 A₂60nmand then the plasmid was diluted to serial standard concentrations for FQ-PCR.The results showed that the target fragment of pIGF-ⅠcDNA 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.更多还原