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目的构建副流感病毒5型(parainfluenza virus 5,PIV5)CC-14株核衣壳蛋白(nucleoprotein,NP)、磷蛋白(phosphoprotein,P)和聚合酶蛋白(large protein,L)基因真核表达质粒,并在MDCK细胞中表达,为该病毒反向遗传系统的建立奠定基础。方法根据PIV5 CC-14株全基因组中的NP、P和L基因分别设计特异性引物,经RT-PCR扩增得到NP、P和L基因,酶切后分别连接至pc DNA3.1+真核表达载体(或p MD18-T)上,构建辅助质粒pc DNA3.1-NP、pc DNA3.1-P和pc DNA3.1-L,转染MDCK细胞,RT-PCR检测NP、P和DL基因的转录情况。结果经双酶切及测序鉴定证明辅助质粒pc DNA3.1-NP、pc DNA3.1-P和pc DNA3.1-L构建正确,3个质粒转染MDCK细胞后,经RT-PCR分别可扩增得到NP、P和DL基因片段。结论成功构建了PIV5 CC-14株pc DNA3.1-NP、pc DNA3.1-P和pc DNA3.1-L辅助质粒,且均可在MDCK细胞中有效转录,为该病毒反向遗传系统的建立奠定了基础。
Objective To construct the nucleoprotein (NP), phosphoprotein (P) and large protein (L) gene eukaryotic expression plasmids of parainfluenza virus 5 (CCV-14) , And expressed in MDCK cells, laying a foundation for the establishment of the reverse genetics system of the virus. Methods According to the NP, P and L genes of PIV5 CC-14 strain, specific primers were designed respectively. The NP, P and L genes were amplified by RT-PCR and ligated into pcDNA3.1 + On the expression vector (or p MD18-T), the helper plasmids pcDNA3.1-NP, pcDNA3.1-P and pcDNA3.1-L were constructed and transfected into MDCK cells. The NP, P and DL genes were detected by RT- The transcription of the situation. Results The DNA plasmids pcDNA3.1-NP, pcDNA3.1-P and pcDNA3.1-L were constructed correctly by double enzyme digestion and sequencing. The three plasmids transfected MDCK cells were amplified by RT-PCR Increased NP, P and DL gene fragments. Conclusion The pcDNA3.1-NP, pcDNA3.1-P and pcDNA3.1-L helper plasmids were constructed successfully and both of them were successfully transcribed in MDCK cells. Establishment laid the foundation.