Objective To Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV. Methods P, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1(+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis. Results Expression of P, NP genes were detected and confirmed by the IE and WB analysis. Conclusion The recombinant eukaryotic plasmids pcDNA3.1 (+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.%目的 构建新城疫病毒LaSota株NP、P基因的真核表达载体,为研究NP、P蛋白机理及反向遗传操作系统奠定基础.方法 利用RT-PCR法扩增出新城疫病毒LaSota株的NP、P基因,并将其克隆到pGEM-T easy载体中,并再次亚克隆到真核载体pcDNA3.1(+)上,得到重组质粒pcDNA3.1(+)-NP、pcDNA3.1(+)-P,瞬时转染到293、BHK-21 细胞中,免疫酶法及Western Blot法检测表达情况.结果 经免疫酶法及Western Blot法分别检测到了P、NP蛋白在两种细胞的表达.结论 构建的两个重组质粒pcDNA3.1(+)-NP、pcDNA3.1(+)-P 在293、BHK-21两种细胞中均可稳定表达,为即将进行的NDV反向遗传操作系统奠定基础.
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