骨唾液蛋白非融合荧光蛋白载体的构建及其在特异性骨转移乳腺癌细胞中的稳定表达

目的 构建骨唾液蛋白(BSP)非融合荧光表达载体，建立稳定高效表达BSP和荧光蛋白的乳腺癌细胞系，为进一步研究BSP在乳腺癌细胞骨转移中的作用奠定基础。方法 从构建好的pB-hBSP载体中用PCR方法扩增hBSP基因，通过BglⅡ和PstⅠ两个限制性酶切位点定向克隆至真核表达载体pIRES2一EGFP，构建重组载体pIRES2-hBSP-EGFP。利用脂质体转染的方法将构建好的重组质粒转入特异性骨转移的乳腺癌细胞株MDA-MB-23180中，并利用G418和流式细胞仪筛选阳性克隆。结果 成功构建hBSP和EGFP非融合表达的真核表达载体pIRES2-hBSP EGFP，并成功转染特异骨转移的乳腺癌细胞株，获得hBSP的稳定转染细胞株，荧光显微镜下可观察到荧光蛋白标记。结论 真核表达载体CRFS2-hBSP-EGFP的构建及hBSP稳定转染细胞株的获得为研究BSP在乳腺癌骨转移中的作用奠定了的基础。

Construction of pIRES2-hBSP-EGFP vector and stably expression in MDA -MB-231BO breast cancer cells

Objective To constitute a higher expression system and to obtain the breast cancer cell strains in which BSP is stably expressed. Methods hBSP gene was subcloned from pB-hBSP vector by PCR. The PCR fragment was inserted into the eukaryon expression vector, pIRES2-EGFP, which allow exogenous protein and EGFP to express respectively. The recombinant vector, pIRES2-hBSP-EGFP, was transfected into human breast cancer cells with Lipofectamine TM 2000. The expression of symbol protein EGFP, could be conveniently observed with fluorescent microscope. Results The recombinant pIRES2-hBSP-EGFP plasmid was constituted and successfully transfected into breast cancer cells. In the breast cancer cell strain hBSP and EGFP were expressed. Conclusion The successful constitution and transfection of hBSP and EGFP nonfusion expression vector laid a foundation for the further study on the effect of BSP on breast cancer metastasizing to bone in vivo or in vitro.