Wnt3a重组真核载体的构建及其在晶状体上皮细胞系SRA01/04细胞的表达

Expression in SRA01/04 cells of lens epithelial cell line and construction of Wnt3a-pcDNA3 recombinated vector

目的构建Wnt3a真核表达载体并检测其在SRA01/04细胞中的瞬时表达情况。方法采用cDNA文库设计的含有Wnt3acDNA全长序列模板的菌种,PCR法获得Wnt3a片段,与pcDNA3连接,进行酶切鉴定及测序。重组质粒瞬时转染人晶状体上皮细胞系SRA01/04细胞,检测Wnt3a蛋白表达情况。结果 Wnt3a-pcDNA3表达载体基因测序与Genebank报告基因序列一致,Wnt3a-pcDNA3瞬时转染至SRA01/04细胞,Wnt3a蛋白表达明显增强;正常人晶状体上皮细胞系SRA01/04中,仅有少量Wnt3a表达。结论成功构建Wnt3a-pcDNA3真核表达,获得Wnt3a/SRA01/04细胞系,为研究后囊膜混浊的病理机制和防治提供实验基础。

Objective To construct the Wnt3a-pcDNA3 recombinated vector and investigate its transient expression in SRA01/04 cells.Methods The bacteria containing Wnt3a cDNA was designed based on cDNA library.Wnt3a gene from PCR was connected with pcDNA3 to construct Wnt3a-pcDNA3 expression vector,which was identified by digestion and genetic sequencing.The Wnt3a-pcDNA3 plasmid was transient transfected into human lens epithelial cells SRA01/04 cells.The expression of wnt3a protein was detected.Results Recombinated Wnt3a-pcDNA3 was obtained and its genetic sequencing was identical with Genebank.The expression of wnt3a protein was increased in SRA01/04 cells tansfected with Wnt3a-pcDNA3 plasmid,which was slightly expressed in normal SRA01/04 cells.Conclusion The successful construction of Wnt3a-pcDNA3 expression vector and Wnt3a/SRA0104 cells provides the experimental foundation for the further study the pathologic mechanism and prevention of posterior capsular opacification.