人血管内皮细胞生长因子基因克隆、表达与纯化

目的研究人血管内皮细胞生长因子121(hVEGF121)基因在pET-24a(+)中的表达,获得高效表达、具有生物学活性的rhVEGF121。方法提取人原髓白血病细胞(HL60)总RNA,RT-PCR扩增hVEGF121基因,经DNA序列分析后,克隆于高效原核表达载体pET-24a(+),并转化到大肠杆菌B121(DE3)中,经IPTG诱导表达,超滤复性,DEAESephamseFastFlow阴离子交换和SephacryS-100分子筛色谱纯化后,以HUVEC测定其生物学活性。结果SDS-PAGE结果显示,目的蛋白质以包涵体形式存在,表达量达菌体总蛋白质的20%,纯化后rhVEGF121纯度达95%以上,生物学活性检测证实,具有刺激HUVEC增殖的功能。结论在原核系统中实现了hVEGF121的高效表达。

The gene cloning, expression and purification of human vascular endothelial growth factor 121

Purpose To study the expression of human vascular endothelial growth factor 121(VEGF121) cDNA in pET-24a(+) and to obtain high-level expressed recombinant human VEGF121 with good biological activity. Methods The total RNA was extracted from HL60 cells and the cDNA encoding hVEGF121 was amplified by using RT-PCR. After being confirmed by DNA sequence analysis, the cDNA was inserted into the prokaryotic expression vector pET-24a(+) and induced by IPTG to express the hVEGF121. The protein was refolded by ultrafiltration and purified by DEAE-Sepherose FF and Sephacry S-100 chromatography,and its biological activity was assayed. Results By SDS-PAGE analysis, the hVEGF121 expressed in E. coli. existed in form of inclusion bodies, and the expressed hVEGF121 came up to 20% of total proteins. The purity of the purified rhVEGF121 reached more than 95% and was proved to have a good biological activity to stimulate HUVEC proliferation. Conclusion rhVEGF121 was highly expressed successfully in E. coli.