重组人色素上皮细胞衍生因子融合蛋白的制备及其抑制新生血管

目的：人色素上皮细胞衍生因子 (pigment epithelium-derived factor, PEDF)是一种有效的新生血管形成抑制因子和神经营养因子。本文通过原核细胞表达人PEDF蛋白，鉴定其抑制新生血管的生物学活性。方法：采用PCR法扩增人PEDFcDNA，将其克隆到pET32a载体中，在大肠杆菌BL21中表达人PEDF蛋白。经SDS-PAGE和Western-blot鉴定后，镍柱亲和层析法变性条件下纯化重组融合蛋白。Bradford法测蛋白浓度，采用鸡胚脲囊膜法测其对新生血管形成的影响。结果：成功构建了pET32a-PEDF表达载体。重组人PEDF蛋白在BL21宿主菌中获得了稳定高效表达，鸡胚脲囊膜实验结果显示在重组蛋白浓度为0.4、0.04 ng/ml时均有对新生血管的显著抑制作用（P<0.01），而在4 ng/ml时无抑制作用。结论： 成功高效表达及纯化了重组人PEDF蛋白，鉴定其抑制新生血管的生物学活性，并且证实该活性在一定范围内有效，为进一步研究其功能及推广应用奠定了基础。

The preparation of the recombinant Fusion protein Human Retinal Pigment Epithelium-Derived Factor and the analysis of its angiogenesis activity

Aim The recombinant Human retinal pigment epithelium-derived factor（PEDF）protein to be obtained and the angiogenesis of the rPEDF to be identified. Method PEDF gene was amplified by PCR and cloned into pET32a, rPEDF protein was expressed in E coli BL21 and confirmed by SDS-PAGE and Western-blot. The rPEDF was purified by Ni-NTA on denature condition. The concentration of the rPEDF was determined by Bradford method. The angiogenesis of the rPEDF was determined by chick chorioallantoic membrane (CAM) method. Results The expression plasmid pET32a-PEDF was constructed successfully .The rPEDF was expressed stably efficiency in E coli BL21. The results of the CAM experiment showed that the rPEDF had notable angiogenesis effect in the concentration 0.4、0.04 ng/ml, but had no effect in 4 ng/ml. Conclusion: The PEDF gene was cloned and expressed efficiency, the angiogenesis of the rPEDF to be identified and the activity was worked in certain range. The results can facilitate studying its function and spreading its application.