_catenin基因真核表达载体的构建及蛋白的表达和定位

解剖科学进展　Progress of Anatomical Sciences　2014 Sep, 20(5): 436~439

β-catenin 基因真核表达载体的构建及蛋白的表达和定位

1211*1

张成宏，沈 涛，佟宇鑫，李 妍，李 丰（中国医科大学 1. 基础医学院细胞生物学教研室 细胞生物学卫生部重点实验室，2. 附属盛京医院骨一科，辽宁 沈阳 110004）

Construction of eukaryotic plasmid of human β-catenin gene and the

expression and localization of its fusion protein

1211*1

ZHANG Cheng-hong , SHEN Tao , TONG Yu-xin , LI Yan , LI Feng （1. Department of Cell Biology, the Key Laboratory of Cell Biology of Ministry of Education and Public Health of China, College of Basic Medical Sciences, and 2. Department of Orthopedic Surgery of Shengjing Hospital, China Medical University, Shenyang 110004, China）

【Abstract】　Objective To construct the expression plasmid of beta-catenin (or β-catenin) gene and identify the expression and localization of its fusion protein. Methods　Total mRNA was extracted from NIH3T3 cells, cDNA was formed by reverse transcription. The β-catenin coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into pEGFP-C1 vector. After the target region was identified by enzyme digestion and sequencing, the plasmid was transfected into NIH3T3 cells. The expression of the recombinant plasmid in NIH3T3 cells was detected by Western blot. The localization of pEGFP-β-catenin in NIH3T3 cells was observed with laser scanning confocal microscopy. Results　The length of the fragment identified by restriction enzyme digestion was 2346bp. The expression of pEGFP-β-catenin fusion protein with a molecular weight of 115kDa was detected by Western blot. The pEGFP-β-catenin fusion protein was mostly localized in the membrane and cytoplasm of NIH3T3 cells. Conclusion　The recombinant plasmid of β-catenin gene was successfully cloned into eukaryotic expressing vector, and the pEGFP-β-catenin fusion protein was mostly localized in the membrane and cytoplasm of NIH3T3 cells.

[Key words]β-catenin; plasmid construction; NIH3T3 cell; gene expression and localization

【摘要】　目的　构建β-catenin真核表达载体并证实融合蛋白在细胞内的表达及定位。方法　提取工具细胞NIH3T3的mRNA，反转录为cDNA。PCR扩增β-catenin基因cDNA全长，并将其亚克隆至pEGFP-C1表达载体中。进一步将构建的重组质粒进行酶切和测序鉴定，并转染到工具细胞NIH3T3细胞中，提取细胞蛋白进行Western blot检测。最后利用激光扫描共聚焦显微镜观察pEGFP-β-catenin在NIH3T3 细胞内的定位。结果　β-catenin基因cDNA全长克隆到了真核表达载体pEGFP-C1中，酶切鉴定片段为2346 bp，并测序成功。Western blot检测到了GFP-β-catenin融合蛋白表达，分子量约为115kDa。pEGFP-β-catenin在工具细胞NIH3T3细胞中主要定位于细胞膜和细胞质。结论　成功构建了β-catenin基因cDNA全长的真核表达载体，pEGFP-β-catenin蛋白在NIH3T3细胞中主要定位于细胞膜和细胞质。

【关键词】　β-catenin基因；质粒构建；NIH3T3细胞；基因表达与定位

【中图分类号】　Q257　【文献标志码】　A　【文章编号】　1006-2947(2014)05-0436-04

【收稿日期】2014-01-05

【基金项目】国家自然科学基金资助项目（81372337）和辽宁省 自然科学基金项目（2013021090）

* 通讯作者 (To whom correspondence should be addressed)

连接蛋白（catenins）是一类与上皮性钙粘素（E-cadherin）细胞内区连接的胞浆蛋白包括α-[12]catenin、 β-catenin、 γ- catenin和p120-catenin等。其中β- catenin是经典的 Wnt信号转导通路的关键蛋[3]白，在胚胎发育和肿瘤发生中起着重要的作用。

，,β-catenin基因位于染色体3p21由 16 个外显子组成，其中cDNA全长2346bp。研究表明β-catenin 是一种多功能蛋白，即是E-cadherin复合体的重要组成部分，调节细胞间的粘附；又是经典Wnt 信号传导通路的关键调控点，介导Wnt信号通路从膜经质进[4,5]核的传递，在胚胎发育及肿瘤发生中起关键作用。

本研究通过构建β-catenin基因cDNA全长的真核表达载体，并证实了其在细胞内的表达与定位，

，