骨形态发生蛋白的可溶性表达、纯化及抗体制备

Soluble Expression,Purification of Bone Morphogenetic Protein-2 Fragment and its Antibody Preparation

目的用RT-PCR技术优化表达条件获得表达量比较高的BMP-2ω蛋白,获得纯度较高的蛋白。方法用RT-PCR技术从SAOS-2细胞的总RNA中扩增出BMP-2基因片段BMP-2ω(606-846bp),并插入到原核表达载体pET-28a(+)上;转化大肠杆菌BL21(DE3),进行诱导表达研究,使其在大肠杆菌中能够可溶性表达,用镍离子螯和柱(Ni-NTA)纯化BMP-2ω蛋白,获得纯度较高的蛋白,用纯蛋白免疫小鼠制备多克隆抗体。结果ELISA结果显示效价可达到1∶6400;Western印迹结果表明该抗体可以与BMP-2蛋白特异结合。结论为BMP-2在骨肉瘤发生、发展中的作用的研究奠定了基础。

The bone morphogenetie protein-2 （BMP-2） fragment （BMP-2ω, 606-846bp） cDNA was amplified from total RNA of SAOS-2 cells by using RT-PCR. The PCR product was then inserted into pET-28a （＋） vector for constructing the expression plasmid that would be used to transform the host cell BL21 （DE3）. After IPTG inducing under different conditions, this BMP-2ω protein could be expressed in high level as a soluble form, and purified by chelating column （Ni-NTA）. Polyelonal antibody was made by immunizing mice with using purified protein, and the antiserum titer generated was 1 ： 6400 that was measured by ELISA. Western blot result showed that this antibody could bind to BMP-2 protein specifically. Above research result set up the basis for studying on the treatment of osteosareoma.