人源血管内皮生长因子突变体Ⅱ的克隆表达、分离纯化及初步鉴定

Cloning,Expression,Purification and Preliminary Identification of Human Vascular Endothelial Growth Factor Mutant Ⅱ

构建人源血管内皮生长因子突变体hVEGF121Ⅱ的重组原核表达质粒,获得hVEGF121Ⅱ蛋白用于制备抗血管生成作用更强的抗肿瘤疫苗。利用PCR技术,在已构建的hVEGF121Ⅰ突变体的基因末端引入热休克蛋白mHSP70的407-426两段串联重复序列的编码基因,PCR产物经Hind III和Nco I双酶切后连接到经同样限制性内切酶切过的原核表达载体pET-28a(+)上,转入大肠杆菌菌株BL21(DE3)中,乳糖诱导表达,超声破碎菌体,包涵体经洗涤、溶解、稀释复性、离子交换层析得到目的蛋白hVEGF121Ⅱ,Western-blot鉴定。结果成功构建了hVEGF121Ⅱ蛋白的表达质粒pET-28a(+)-hVEGF121Ⅱ,重组菌株经乳糖诱导后,目的蛋白表达量占全菌蛋白的62%,分离纯化后纯度达到95%,hVEGF121Ⅱ蛋白的单体和二聚体都可以和VEGF抗体结合。重组hVEGF121Ⅱ蛋白的表达成功为进一步对其进行免疫学研究奠定了基础。

To obtain the hVEGF121 I1 protein for the preparation of anti-tumor vaccines which were stronger to resist angiogenesis, the recombinant prokaryotie plasmid expressing human vascular endothelial growth faetor mutant hVEGF121 I1 was constructed. By PCR technique, the gene encoding two tandem repeat sequences of peptide 407-426 inside heat shock protein mHSP70 was intro- duced to the terminus of the constructed hVEGF121 I mutant gene. PCR product digested with Hind III and NcoI was connected to the prokaryotic expression vector pET-28a （ ＋ ） digested with the same restriction endonuclease. Then the recombinant plasmid was transformed into E. coli strain BL21 （DE3）. After the reorganized strain was induced with lactose and lysised by ultrasonic, the resulting inclusion body was washed, dissolved, and diluted to renature and purified by ion-exchange chromatography to get the target protein hVEGF121 I1 ,and then it was identified by Western-blot. The recombinant plasmid pET-28a（ ＋ ）-hVEGF121 II expressing hVEGF121 II was successfully constructed. After the recombinant strains were induced with galactose, the target protein accounted for 62% of the whole bacteria cell proteins ,and its purity reached 95% after separation and purification procedure. Both the monomer and dimmer of the hVEGF121 II protein could eombine with VEGF antibody. The successful expression of hVEGF121 II protein has laid the foundation for its immunology research.