绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定

doi:10.11843/j.issn.0366-6964.2017.04.006

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (4): 637-644.doi: 10.11843/j.issn.0366-6964.2017.04.006

绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定

马亚茹¹, 胡广东², 王红红¹, 加米拉², 徐锦凤¹, 陈创夫^2*, 赛务加甫^2*

1. 石河子大学生命科学学院, 石河子 832003;
2. 石河子大学动物科技学院, 石河子 832003

收稿日期:2016-10-25 出版日期:2017-04-23 发布日期:2017-04-21
通讯作者: 陈创夫，博士，教授，主要从事畜禽病原分子生物学研究，E-mail：ccf-xb@163.com；赛务加甫，博士，教授，主要从事转基因动物研究，E-mail：291016059@qq.com
作者简介:马亚茹（1990-），女，陕西合阳人，硕士生，主要从事动物基因工程研究，E-mail：861228697@qq.com
基金资助:
国家自然科学基金项目（31460683）

Construction and Identification of BHK-21 Cell Line Stably Expressing Ovine NYD-SP27

MA Ya-ru¹, HU Guang-dong², WANG Hong-hong¹, JIA Mi-la², XU Jin-feng¹, CHEN Chuang-fu^2*, SAI Wu-jia-fu^2*

1. College of Life Sciences, Shihezi University, Shihezi 832003, China;
2. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China

Received:2016-10-25 Online:2017-04-23 Published:2017-04-21

摘要/Abstract

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63 ku；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。

关键词: 绵羊NYD-SP27基因, 稳定表达, BHK-21细胞, P2A

Abstract:

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 pDsRed1-C1. To co-express Red fluorescent protein 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 immunofluorescence 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 fluorescent protein. The eukaryotic expression vectors were confirmed successfully and the BHK-21 cell lines stably expressing ovine NYD-SP27 was successfully established in this study.

Key words: ovine NYD-SP27 gene, stably expressing, BHK-21 cells, P2A

中图分类号:

S826.2

马亚茹, 胡广东, 王红红, 加米拉, 徐锦凤, 陈创夫, 赛务加甫. 绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定[J]. 畜牧兽医学报, 2017, 48(4): 637-644.

MA Ya-ru, HU Guang-dong, WANG Hong-hong, JIA Mi-la, XU Jin-feng, CHEN Chuang-fu, SAI Wu-jia-fu. Construction and Identification of BHK-21 Cell Line Stably Expressing Ovine NYD-SP27[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(4): 637-644.

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绵羊NYD-SP27基因在BHK-21细胞中的稳定表达及其特征鉴定

Construction and Identification of BHK-21 Cell Line Stably Expressing Ovine NYD-SP27

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