Position: Home > Articles > Construction of Eukaryotic Expression Vector and Sub-cellular Location of Suqin Yellow Chicken Homolog to Vasa Gene(CVH)
Chinese Journal of Animal Science
2014,50
(9)
82-87
苏禽黄鸡CVH基因真核表达载体构建及亚细胞定位
作 者：
朱睿;张振韬;左其生;刘志永;韦光辉;李东;连超;张亚妮;李碧春
单 位：
扬州大学动物科学技术学院
关键词：
CVH基因;亚细胞定位;EGFP;间接免疫荧光;多克隆抗体;黄鸡
摘 要：
本研究通过PCR技术克隆苏禽黄鸡CVH基因的CDS区,分别构建真核表达载体pcDNA3.1-CVH和pEGFP-C1-CVH,并用pcDNA3.1-CVH载体免疫小鼠制备多克隆抗体。pcDNA3.1-CVH和pEGFP-C1-CVH分别转染COS-7细胞,进行间接免疫荧光实验和CVH-EGFP融合蛋白亚细胞定位,并通过RT-PCR、Western blot进一步检测蛋白的表达。结果表明:克隆出CVH基因的完整CDS,构建了真核表达载体pcDNA3.1-CVH和pEGFP-C1-CVH,成功制备了多克隆抗体,转染后观察CVH基因表达产物定位于细胞质中,RT-PCR和Western bolt分别检测到1 989 bp特异条带和100 ku的融合蛋白。真核表达载体制备的多克隆抗体特异性良好,效价为1∶200,CVH基因蛋白在细胞质中表达,为进一步探讨CVH基因的生物学特性建立基础。
译 名：
Construction of Eukaryotic Expression Vector and Sub-cellular Location of Suqin Yellow Chicken Homolog to Vasa Gene(CVH)
作 者：
ZHU Rui;ZHANG Zhen-tao;ZUO Qi-sheng;LIU Zhi-yong;WEI Guang-hui;LI Dong;LIAN Chao;ZHANG Ya-ni;LI Bi-chun;Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province,College of Animal Science& Technology,Yangzhou University;
关键词：
CVH gene;;subcellular localization;;EGFP;;indirect immunofluorescence assay;;multiclonal antibody
摘 要：
The aims of the study were to clone cDNA of chicken homolog to Vasa gene(CVH),and prepare multiclonal antibody against CVH using of eukaryotic expression vector,then analyze the sub-cellular localization of the expression product through both EGFP fusion protein and indirect immunofluorescent assay in the transfected COS-7cell.CVH gene was amplified by PCR from cDNA of chicken and inserted into the eukaryotic expression vector pcDNA3.1 and pEGFP-C1 to construct eukaryotic expression vector pcDNA3.1-CVH,which was used for mouse immune to prepare multiclonal antibody against CVH,and fusion protein expression vector pEGFP-C1-CVH.The two vectors were transfected into COS-7 cells,respectively,then CVH protein expression was detected in COS-7 cells by indirect immunofluorescent assay and EGFP-CVH fusion protein.Then mRNA and protein expression in vitro was detected using RT-PCR and Western blot methods.The cDNA of CVH gene was successfully cloned,and the eukaryotic expression vectors pcDNA3.1-CVH and pEGFP-C1-CVH were constructed successfully;the multiclonal antibody against CVH had been prepared successfully.EGFP-CVH fusion protein was located in the cytoplasmic of COS-7 cells,and so was CVH protein of pcDNA3.1-CVH.RT-PCR detected specific 1 989 bp hand,indicating the expression of fusion protein in RNA level.Western blot analysis confirmed that EGFP-CVH fusion protein of Mr 100kD was detected in transfected COS-7 cells.The multiclonal antibody against CVH had been prepared successfully;Serum antibody titer is 1 ∶200;the CVH protein was efficiently expressed in the cytoplasm of COS-7 cells.These results had a good value for further research of CVH function.
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