摘要
旨在构建稳定表达绵羊NYD-SP27基因的BHK-21细胞株,对NYD-SP27基因的表达特征进行分析,为进一步研究NYD-SP27基因功能奠定基础。本研究克隆绵羊NYD-SP27基因,并将其插入到真核表达载体pDsRed1-C1中,利用猪捷申病毒2A肽(P2A)将NYD-SP27蛋白和红色荧光蛋白连接以实现共表达,将重组质粒pDsRed-P2A-Flag-NYDSP转染至BHK-21细胞中,经G418筛选获得稳定转基因细胞株,利用RT-PCR、间接免疫荧光(Indirect immunofluorescence assay,IFA)和Western blot对NYD-SP27基因的表达进行鉴定。结果表明,NYD-SP27基因稳定整合至宿主细胞基因组;NYD-SP27蛋白能够在BHK-21细胞中正常表达,分子大小约为63ku;在NYD-SP27蛋白和红色荧光蛋白翻译过程中,P2A成功自我剪切。本研究成功构建了绵羊NYD-SP27基因的真核表达载体,建立了稳定表达绵羊NYD-SP27基因的BHK-21细胞系。
In order to study function of ovine NYD-SP27,BHK-21 cell line stably expressing ovine NYD-SP27 was constructed and relative phenotypic characteristics were analyzed, which lay a foundation for function analysis of ovine NYD-SP27 gene. The ovine NYD-SP27 gene was cloned and inserted into eukaryotic expressing vector pDsRedl-C1. To co-express Red fluorescent pro- tein gene and NYD-SP27 gene, both of them were linked with porcine teschovirus-1 2A peptide （P2A）. The recombinant plasmid pDsRed-P2A-Flag-NYDSP was transfected into BHK-21 cells and selected with G418. Positive cells were identified using RT-PCR, indirect immunofluores- cence assay （IFA） and Western blot. The RT-PCR results indicated that NYD-SP27 gene was stably integrated into genome of BHK-21 cells. The NYD-SP27 protein could be expressed in BHK-21 cell line and the molecular weight was approximately 63 ku, which suggested that the self-cleavage of P2A realized during the translation process of NYD-SP27 protein and Red fluores- cent protein. The eukaryotic expression vectors were confirmed successfully and the BHK-21 celllines stably expressing ovine NYD-SP27 was successfully established in this study.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第4期637-644,共8页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金项目(31460683)
