苏禽黄鸡CVH基因真核表达载体构建及亚细胞定位  

Construction of Eukaryotic Expression Vector and Sub-cellular Location of Suqin Yellow Chicken Homolog to Vasa Gene(CVH)

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 

机构地区：[1]扬州大学动物科学技术学院,江苏省动物遗传繁育与分子设计重点实验室,江苏扬州225009

出　　处：《中国畜牧杂志》2014年第9期82-87,共6页Chinese Journal of Animal Science

基　　金：国家自然基金(31272429);江苏省研究生培养创新工程(CXLX12_0934)

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。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-7 cell. CVH gene was amplified by PCR from eDNA 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 eDNA 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 100 kD 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.

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 

分 类 号：S831.2[农业科学—畜牧学]