鸡Piwi蛋白的亚细胞定位研究  

Sub-cellular Localization of Chicken Piwi Protein

作　　者：戴爱琴[1] 常国斌[1] 陈蓉[1] 张颖[1] 阴彦辉[1] 马腾[1] 李建超[1] 陈国宏[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2013年第1期15-22,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31172199;30972088);江苏省自然科学基金(BK2009190);江苏高校优势学科建设工程资助项目

摘　　要：通过构建2种真核表达载体比较Piwi蛋白在真核细胞中的表达部位,为进一步研究Piwi蛋白的生物学功能提供依据。采用RT-PCR方法克隆了狼山鸡睾丸组织中Piwi基因CDS,构建含有增强型绿色荧光蛋白(EGFP)报告基因的融合表达载体pEGFP-C1-Piwi,脂质体PEI介导基因转染NIH-3T3细胞,同时构建pcDNA3.1-Piwi真核表达载体,采用细胞间接免疫荧光方法检测Piwi蛋白在NIH-3T3细胞的表达部位。结果表明,成功克隆出公鸡Piwi基因的全序列CDS,大小为2 604bp,与GenBank中预计的序列基本一致;融合表达载体pEGFP-C1-Piwi在整个细胞中都有表达;而间接免疫荧光方法检测到pcDNA3.1-Piwi载体主要在NIH-3T3细胞质中表达。Piwi蛋白主要在细胞质中表达,采用间接免疫荧光方法比EGFP报告基因法更为可靠,适合目的蛋白的亚细胞定位研究。The localization of Piwi protein in eukaryotic cells was conducted by constructing two eukaryotic expression vectors, aimed to provide basis for further study on the biological functions of Piwi protein. CDS of Piwi gene from Langshan chicken testicular tissue was cloned by Reverse Transcript-PCR. Fusion expression vector containing the enhanced green fluorescent protein （EGFP）, pEGFP-C1-Piwi, was constructed; then was transfected into NIH-3T3 with Lipofectin PEI. Meanwhile, the eukaryotic expression vector pcDNA3.1-Piwi was also constructed success- fully, and then Piwi protein expression was detected in NIH-3T3 ceils by indirect immunofluores- cence assay. CDS of Piwi gene was cloned successfully, and the sequence size was 2 604 bp, which was similar with GenBank. Fusion expression vector pEGFP-C1-Piwi expressed in the whole NIH-3T3 cells, while Piwi protein of pcDNA3.1-Piwi was found to locate in the cytoplas- mic of NIH-3T3 ceils by indirect immunofluorescence assay. Piwi protein mainly expressed in the cytoplasmic of NIH-3T3 cells, and indirect immunofluorescence assay was more reliable than the EGFP reporter gene method, which was fit for investigating sub-cellular localization of target protein.

关 键 词：Piwi蛋白 真核表达 亚细胞定位 绿色荧光蛋白 间接免疫荧光 

分 类 号：S831.2[农业科学—畜牧学]