猪GM-CSF稳定表达细胞系的建立

doi:10.11843/j.issn.0366-6964.2017.05.022

Abstract

Abstract:

This study was conducted to establish cells lines stably expressing porcine GM-CSF. The primer of porcine GM-CSF was designed according to the sequence published in NCBI, the open reading frame (ORF) was cloned by RT-PCR method. The porcine GM-CSF gene cloned consisted of 435 bp, and the ORF of porcine GM-CSF encoded a protein of 144 amino acids. The GM-CSF polypeptide had an estimated molecular mass of 16.3 ku, PI of 7.15 and displayed a high homology to Ovis aries(77.1%), Equus caballus (73.6%), Felis catus(72.2%), Bos taurus(71.5%), Canis lupus familiaris(71.3%)and Homo sapiens(70.8%), respectively. After the digestion of pIRES2-EGFP vector by EcoR I and BamH I enzyme, the ORF of porcine GM-CSF was inserted and named pIRES2-EGFP-pGM-CSF. Following, the pIRES2-EGFP-pGM-CSF was transfected into porcine intestinal epithelial cell line IPEC-J2, which was then screened by G418. Consequently, the expression of GM-CSF mRNA in transfected cells was significantly increased compared to control cells and after 24 h of transfection(P<0.01), and transfected cells also showed high expression of green fluorescent. After G418 selection and expansion (positive clone) for 30 passages, the expression of GM-CSF mRNA was increased to control compared cells. We concluded that the eukaryotic expression vector pIRES2-EGFP-pGM-CSF was constructed successfully and a cell lines that show the stable expression of porcine GM-CSF was established.

CLC Number:

S826.2

LI Jun-min, YONG Yan-hong, GONG Dong-liang, FENG Yuan, WEI Xuan, HE Yu-lin, JU Xiang-hong. The Establishment of a Cell Line Stably Expressing Porcine GM-CSF[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 963-970.

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The Establishment of a Cell Line Stably Expressing Porcine GM-CSF

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