天花粉蛋白的盐析工艺和亲和双水相分离工艺

Salting-out process and affinity aqueous two-phase extraction process of trichosanthin

近年,随着天花粉蛋白(TCS)在抗癌细胞和HIV病毒方面的临床性研究进展,天花粉蛋白生产工艺也需在保证效益的前提下精细化。本实验利用硫酸铵分级沉淀、硫酸铵粗提后亲和双水相进一步纯化,得到精制的天花粉蛋白。并对提取工艺进行了优化,最后利用SDS聚丙烯酰胺凝胶电泳对其纯度进行了鉴定以及相对分子质量的测定,并对比了盐析法和亲和双水相分离法,研究结果表明:硫酸铵沉淀法最佳分离条件为固液比为1∶12、浸泡时间为24h、pH在5左右,最终得率0.8mg/g;亲和萃取最佳条件:染料CibacronBlue F3GA-吐温80在成相吐温80中的含量8%,成相吐温80的占双水相含量为4.0%,(NH4)2SO4的含量2.0mol/L,充分混合的时间9h,反萃取氯化钠的含量为0.5mol/L,反萃取的时间为8h,得率0.6mg/g,但凝胶电泳显示其纯度较高。

In recent years,clinical studies showed that trichosanthin＇s has anti-cancer and anti-HIV virus function.The trichosanthin＇s production needs to be studied to meet the market needs.This experiment used two steps of trichosanthin extraction a） purification trichosanthin with ammonium sulfate precipitation,b） affinity aqueous two-phase extraction after ammonium sulfate coarse separation.The extraction process was optimized and gel electrophoresis was used in identifying the purity and molecular weight.The salting-out method and affinity aqueous two-phase separation method were compared.The best conditions for salting-out separation were： solid-liquid ratio 1∶12,soaking time 24 hours,pH 5,and the yield 0.8ml/g.The best affinity aqueous two-phase separation conditions were： the percentage of Cibacron Blue F3GA-Tween 80 was 8%,the content of Tween 80 in aqueous two-phase was 4.0%,content of（NH4）2SO4 was 2.0mol/L,mixing time 9h,stripping sodium content 0.5mol/L,the anti-extraction time 8h,yield was 0.6mg/g.The electrophoresis showed the products had a high purity.