目的  构建重组白喉毒素（diphtheria toxin，DT）与血管内皮生长因子（vascular endothelial growth factor，VEGF）融合蛋白，研究其对裸鼠肿瘤模型的治疗效果。方法  采用PCR扩增法从人外周血淋巴细胞cDNA中获取编码含8-110位氨基酸片段的VEGF_8-110基因，并从DT基因中扩增得到编码含1-384位氨基酸片段的DT₃₈₄基因，将目的基因克隆到质粒载体pET9a中，构建重组表达质粒，转化大肠埃希菌BL21-Plys菌株表达重组蛋白，通过阴离子交换层析和分子筛层析纯化。将滴有融合蛋白的滤纸放到暴露的鸡胚绒毛尿囊膜上，孵育24 h后，观察融合蛋白对鸡胚绒毛尿囊膜的影响。对接种肿瘤1周后的裸鼠进行尾静脉注射治疗，第21天处死裸鼠取下肿瘤，测量其体积和质量，观察融合蛋白对裸鼠肿瘤模型的治疗效果。 结果  重组质粒经测序鉴定构建正确。融合蛋白在大肠埃希菌中以包涵体形式表达，包涵体具有高效的复性效果。DT₃₈₄-VEGF_8-110融合蛋白无法抑制鸡胚绒毛尿囊膜上的血管生长，DT₃₈₄-（VEGF_8-110）₂融合蛋白可以抑制鸡胚绒毛尿囊膜上的血管生长，也可抑制裸鼠肿瘤生长。经DT₃₈₄-(VEGF_8-110)₂融合蛋白治疗接种Bxpc3胰腺癌细胞的裸鼠，其肿瘤平均质量（t=5.908，P<0.001）和平均肿瘤体积（t=4.619，P<0.001）均小于对照组。 结论  DT₃₈₄-（VEGF_8-110）₂融合蛋白能够有效抑制裸鼠肿瘤生长。

Objective  To construct fusion protein of diphtheria toxin（DT） and vascular endothelial growth factor（VEGF）, and to study the therapeutic effect of fusion protein on nude mouse tumor model. Methods  Using PCR amplification, VEGF_8-110 gene containing 8－110 amino acid fragments were obtained from peripheral blood lymphocyte cDNA，and DT₃₈₄ gene containing 1－384 amino acid fragments was obtained from DT. The gene fragments were cloned into pET9a to construct recombinant expression plasmids, then transformed into E.coli BL21-plys to express recombinant proteins. Recombinant proteins were purified by ion exchange chromatography and gel chromatography. Filter paper soaked with fusion protein was placed on exposed chick chorioallantoic membrane(CAM) for 24 h incubation. The immunotoxins effect on CAM was observed. Nude  mice inoculated with tumor for one week were treated by tail vein injection of fusion protein. Mice were killed and  tumors were removed at 21 d to measure the volume and mass. The treatment effect on nude mouse tumor was  observed. Results  Sequencing proved that recombinant plasmids were constructed correctly. Fusion proteins were expressed in the form of inclusion body in E.coli. Inclusion bodies had high renaturation efficiency. DT₃₈₄-VEGF_8-110 did not inhibit the blood vessel growth of CAM, but DT₃₈₄- (VEGF_8-110) ₂ inhibited the blood vessel growth of CAM as well as the tumor growth in nude mouse. After 3 weeks of treatment with DT₃₈₄- (VEGF_8-110) ₂ fusion protein in nude mice inoculated with Bxpc3 pancreatic cancer cells, the average tumor mass of the experimental group (t = 5.908, P < 0.001) and the mean tumor volume(t = 4.619, P < 0.001) was both significantly smaller than control group.  Conclusion  DT₃₈₄-（VEGF_8-110）₂  fusion protein can effectively inhibit the tumor growth of nude mouse.