大鼠抗小鼠重组Foxc2单克隆抗体的制备与检测应用

目的：为了研究叉头框c2(Foxc2)在骨骼和主动脉发育上的作用。方法：利用基因重组融合蛋白制作大鼠抗小鼠Foxc2单克隆抗体(抗-Foxc2 mAb)，并利用蛋白质印迹和免疫荧光分析了Foxc2蛋白在小鼠肌胚细胞C2C12的表达和在小鼠胚胎的表达部位。结果：用小鼠GST-Foxc2融合蛋白免疫大鼠制得的抗-Foxc2 mAb效价为1：5000，并特异地与转染了CX-Foxc2质粒的小鼠L细胞和人膀胱癌HTB9细胞产生的Foxc2蛋白结合。在骨成形蛋白-2(BMP-2)的诱导下，Foxc2蛋白在C2C12细胞中显著地增加，用反义Foxc2序列转染C2C12细胞可以明显地抑制Foxc2蛋白在这个细胞系中的表达。Foxc2蛋白表达主要定位于11．5-dpc小鼠胚胎的生骨节和主动脉周围的间充质组织。结论：应用大鼠抗小鼠基因重组Foxc2单克隆抗体研究Foxc2的作用，结果表明Foxc2蛋白在BMP-2的诱导下，在调节C2C12细胞向成骨细胞分化以及在胚胎中轴骨骼和心血管的形成过程中起重要作用。

Preparation and detection application of rat mAb against recombinant mouse Foxc2

Objective:In order to study the effect of forkhead box c2 (Foxc2) on the development of skeleton and aorta Methods:Rat monoclonal anti mouse Foxc2 antibody was prepared by using recombinant fusion protein GST Foxc2, and used it to detect the expression of Foxc2 protein in C2C12 myoblasts and embryo of mouse Results:The titer of rat anti mouse Foxc2 antibody was 1: 5 000 This monoclonal antibody can specifically recognized Foxc2 protein producing in mouse L cells and human bladder carcinoma HTB9 cells transfected with CX Foxc2 plasmid Endogenous Foxc2 protein was detected in the mouse myoblast C2C12 cells and increased significantly in C2C12 cells after treatment with bone morphogenetic protein 2(BMP 2) Endogenous Foxc2 protein level was lowered markedly by transfecting C2C12 cells with antisense Foxc2 sequence Foxc2 protein in 11 5 dpc embryo was localized in the developing selerotome and mesenchymal tissue around the aorta Conclusion:Rat anti mouse Foxc2 monoclonal antibody was successfully prepared by using recombinant fusion protein GST Foxc2 The BMP 2 induced Foxc2 protein may play an important role in regulating the osteoblastic differentiation of C2C12 myoblasts and in the formation of axial skeleton and aortic arch