| pCDNA3-PTH真核表达载体构建及其在细胞内表达 |
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| 引用本文： | 俞路,章世元,王雅倩,刘波,李治学,周联高,严桂芹,汪益峰,林显华.pCDNA3-PTH真核表达载体构建及其在细胞内表达[J].畜牧与兽医,2008,40(10). |
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| 作者姓名： | 俞路 章世元 王雅倩 刘波 李治学 周联高 严桂芹 汪益峰 林显华 |
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| 作者单位： | 扬州大学动物科学与技术学院,江苏,扬州,225009 |
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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。
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| 关 键 词： | 甲状旁腺素 转染 间接免疫荧光试验(IFA) |
Construction of eukaryotic expressing plasmid of chicken PTH gene and its expression in cells |
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| YU Lu,ZHANG Shi-yuan,WANG Ya-qian,LIU Bo,LI Zhi-xue,ZHOU Lian-gao,YAN Gui-qin,WANG Yi-feng,LIN Xian-hua.Construction of eukaryotic expressing plasmid of chicken PTH gene and its expression in cells[J].Animal Husbandry & Veterinary Medicine,2008,40(10). |
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| Authors: | YU Lu ZHANG Shi-yuan WANG Ya-qian LIU Bo LI Zhi-xue ZHOU Lian-gao YAN Gui-qin WANG Yi-feng LIN Xian-hua |
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| Abstract: | 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 nucleotide 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. |
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| Keywords: | parathyroid hormone transfection IFA |
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