摘要
目的：为获得大量人源血管内皮生长因子突变体（hVEGF121’）的重组蛋白，本文研究了其发酵条件，分离纯化方法．并初步考察了其生物活性。方法：应用PCR介导的定点突变方法，将hVEGF121中对其活性影响较小的1-8位和115～12l位两个肽段替换成破伤风毒素（TT）的两个强表位肽段618-627位和831-837位，构建高效表达的pET28a／hVEGF121’重组质粒，考察诱导条件对目的蛋白表达量的影响；所得包含体经过DEAE-Cellulose离子交换柱纯化；初步比较了VEGF121和突变体VEGF121’的免疫和杭肿瘤效果。结果：最佳表达条件为：乳糖诱导时间6h，诱导浓度2mmol／L；离子交换纯化后可得到电泳纯的突变体hVEGF121’；初步的动物免疫发现突变体VEGF121’和VEGF121有相近的免疫滴度，但前者杭肿瘤效果更显著。结论：经发酵纯化后得到重组人源血管内皮生长因子突变体（hVEGF121’）的纯品，并初步展现良好的抗肿瘤效果.
Aim： To optimize the expression condition of reshaping recombined human vascular endothelial growth factor （hVEGF） and perform the purification and the activity determination. Methods： A couple of primers were designed for PCR uses to displace less influential sequences in human VEGF121 terminus 1 -8 and 115 - 121 with tetanus toxin sequence 618 - 627 and 831 - 837 that could elicit strong response to human immune system. The amplified fragment was inserted into pET28a and then transformed into E. coli BL21. Conditions for lactose induction such as concentration and time were also examined. The product was expressed in inclusion body and was purified with DEAECellulose ion exchange chromatography. Furthermore, antigenicity and antitumor activity were compared to VEGF121. Results： 6 h induction by lactose of 2 mmol/L led to optimized expression of the recombinant protein. The product was purified by DEAE-Cellulose ion exchange chromatography. Though no significant immunogenicity between VEGF121 and VEGF121＇ were detected by ELISA, VEGF121＇ was found to significantly inhibit the growth of S180. Conclusion： Recombined mutant human VEGF121 was successfully expressed and purified. It shows the modest capability of inhibition of S180 growth.
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2006年第3期273-276,共4页
Journal of China Pharmaceutical University
