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pCDNA3-PTH真核表达载体构建及其在细胞内表达

Construction of eukaryotic expressing plasmid of chicken PTH gene and its expression in cells
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摘要 以鸡甲状旁腺组织中总RNA为模板,采用RT-PCR法获得甲状旁腺素(PTH)cDNA。将该基因克隆至pCDNA3载体中,构建真核细胞表达载体pCDNA3-PTH,DNA测序证明获得了PTH基因,其序列与GenBank报道序列比较,核苷酸同源性为99.4%,在第159位处存在C→A的有意义突变。将测序正确的PTH基因在多聚乙肽亚胺(PEI)介导下转染COS-1细胞,通过间接免疫荧光试验(IFA)检测到了PTH在COS-1中的表达。PTH基因克隆和表达均获得成功,为进一步探讨该基因在改善蛋壳质量中的应用奠定了基础。 Total RNA was extracted from chicken parathyroid gland tissue, and then the parathyroid hormone (PTH) cDNA was obtained by RT-PCR. The PTH gene fragment was cloned into pCDNA3 vector, and thus the pCDNA3-PTH vector was constructed. The PTH gene encoding 119 amino acids was 360 bp in length, and the homologous comparison showed 99.4% with the sequence reported in GenBank (M36522. 1 ), except that one nueleotide changed from C to A at 159 base site. The recombinant plasmid was transfected into COS-1 cells. At 72 hours after transfection, the expression of PTH was confirmed by indirect immunofluorescence assay (IFA) with the antiserum of PTH expressed in COS-1. This study laid a foundation for further applications of PTH in diagnosis and improving eggshell quality.
出处 《畜牧与兽医》 北大核心 2008年第10期1-4,共4页 Animal Husbandry & Veterinary Medicine
关键词 甲状旁腺素 转染 间接免疫荧光试验(IFA) parathyroid hormone transfection IFA
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