隐球菌免疫相关的甘露糖受体MR重组腺病毒载体的构建及鉴定

Construction and identification of recombinant adenovirus of mannose receptor gene

目的从巨噬细胞系RAW264.7基因组中扩增甘露糖受体(MR)基因,克隆至穿梭质粒后包装重组腺病毒,以进一步研究甘露糖受体MR基因对树突状细胞参与抗新生隐球菌免疫的影响。方法采用PCR方法以及基因重组方法扩增并克隆巨噬细胞基因组中的MR基因,包装能表达MR蛋白的重组腺病毒。结果从巨噬细胞基因组获得MR全基因,克隆至pShuttle-CMV载体,包装了MR的重组腺病毒AD-MR,并在HEK293细胞中获得了表达。结论成功克隆巨噬细胞MR基因并构建了可表达MR基因的重组腺病毒载体,为进一步研究MR基因在树突状细胞参与新生隐球菌免疫中的作用奠定基础。

Objective To clone and construct recombinant adenovirus of mannose receptor （MR） gene from RAW264.7 macro- phage cell line. Methods PCR technique and recombination methods were used in cloning and construction recombinant adenovirus of MR gene, Western blot was used to verify the MR protein expressed in HEK293. Results MR gene was amplified from genomic DNA of macrophage and cloned into pShuttle-CMV vector. The linearized pShuttle-CMW plasmid was cotransformed with the pAdEasy-1 vector into the BJ5183 ceils, producing the recombination adenovirus which can express the MR protein. Conclusion Successful cloning of MR gene and construction of MR gene expression recombinant adenovirus will be useful for the further research on the functions of MR in Dendritic ceils.