副流感病毒5型CC-14株辅助质粒的构建及鉴定  

Construction and identification of helper plasmids for parainfluenza virus type 5 strain CC-14

作　　者：高菽蔓 靳红亮 马嫄[1] 严妍[1] 扈荣良[1] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林长春130122

出　　处：《中国生物制品学杂志》2016年第6期588-591,共4页Chinese Journal of Biologicals

摘　　要：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 eukaryotic expression plasmids containing the nucleoprotein（NP）, phosphoprotein（P）and large protein（L）genes of parainfluenza virus type 5（PIV5）strain CC-14, and express in MDCK cells in order to lay a foundation of development of reverse genetic system of the virus. Methods Primers for the NP, P and L genes were designed in accordance with the complete gene sequence of PIV5 CC-14 strain, with which the genes were amplified respectively by RT-PCR and inserted into vector pc DNA3. 1 ＋ or p MD18-T. The constructed recombinant plasmids pc DNA3. 1-NP, pc DNA3. 1-P and pc DNA3. 1-L were transfected to MDCK cells, and the transcriptions of NP, P and DL genes were determined by RT-PCR. Results Restriction analysis and sequencing proved that recombinant plasmids pc DNA3. 1-NP, pc DNA3. 1-P and pc DNA3. 1-L were constructed correctly. NP, P and DL genes were detected by RTPCR in the MDCK cells transfected with the plasmids. Conclusion Three helper plasmids pc DNA3. 1-NP, pc DNA3. 1-P and pc DNA3. 1-L were constructed successfully and expressed effectively in MDCK cells, which laid a foundation of development of reverse genetic system of PIV5.

关 键 词：副流感病毒5型 核衣壳蛋白 磷蛋白 聚合酶蛋白 辅助质粒 MDCK细胞 

分 类 号：R373.13[医药卫生—病原生物学] Q782[医药卫生—基础医学]