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

Abstract

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.

CLC Number:

S826.2

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

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