【摘要】 目的:构建副黏病毒Tianjin株NP基因的真核表达载体,转染HEK293细胞,经G418筛选出稳定表达NP蛋白的细胞。方法:应用RT-PCR技术克隆副黏病毒Tianjin株NP基因,插入真核表达载体pcDNA3.1(+)。经PCR、酶切和测序鉴定后,将构建的pcDNA3.1(+)-NP用LipofectamineTM2000转染试剂转染HEK293细胞。应用免疫荧光法及Western Blot检测NP蛋白的瞬时和稳定表达。结果:RT-PCR扩增得到NP基因,重组质粒pcDNA3.1(+)-NP转染HEK293细胞后,经免疫荧光法检测到目的蛋白的瞬时和稳定表达。WesternBlot法可见NP蛋白瞬时和稳定表达的条带。结论:副黏病毒Tianjin株NP基因在HEK293细胞可以瞬时和稳定表达,为研究副黏病毒Tianjin株NP蛋白功能与病毒致病性、宿主亲嗜性奠定了基础。更多还原

【Abstract】 Objective:To construct eukaryotic expression vector for nucleoprotein gene of paramyxovirus Tianjin strain,transfect HEK293 cells,and screen the stable expression NP protein by neomycin(G418).Methods:The NP gene was cloned by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.1(+).After identifying with restriction enzyme digestion,PCR and sequencing,the recombinant plasmid was transfected into HEK293 cells by lipofectamineTM2000 reagent.The transient and stable expression of target protein was detected by immunofluorescence assay and western blotting.Results:NP gene could be amplified by RT-PCR method and the transient and stable expression of target protein could be detected by immunofluoresence assay and western blotting in HEK293 cells which were transfected with constructed vector.Conclusion:This study constructed pcDNA3.1(+)-NP vector and gained the NP protein stable expression cells.It can set up a base for further studies on NP protein function,the pathogenicity and affinity of paramyxovirus Tianjin strain.更多还原