摘要
为探讨重组鸡白介素18(mChIL-18)毕赤酵母工程菌在5L发酵罐中大规模发酵的工艺及其对新城疫疫苗的免疫增强作用,复苏工程菌GS115/pPIC9K-mChIL-18于YPD培养基,在其D600值达到5.2左右时转入发酵罐中,采用分批补料方式对毕赤酵母工程菌进行高密度发酵。控制和优化各种发酵条件,经历84h结束发酵。将发酵液离心,用6×His镍柱对上清中的表达产物进行纯化,并将纯化后的mChIL-18用脂质体包被后和新城疫疫苗一起对鸡群免疫。结果显示,毕赤酵母工程菌在5L发酵罐采用甲醇诱导补料批式发酵,在pH5.5,溶氧值20%～30%,温度28℃,诱导72h,mChIL-18的表达产量为560mg/L,发酵上清经纯化后纯度可达70%以上;用0.2mL的mChIL-18脂质体能够显著增强机体的免疫力,其中HI抗体效价和淋巴细胞亚群CD4+、CD8+含量均显著高于对照组。这表明mChIL-18毕赤酵母工程菌在5L发酵罐高密度发酵成功,并能显著增强新城疫疫苗的免疫效果,为IL-18在生产实践得到更好的应用奠定了基础。
To study large-scale fermentation of mature chicken interleukin-18 （mChlL-18） in P. pastoris and its immu noenhancement on Newcastle disease vaccine. The strain GS115/pPIC9K-mChIL 18 was inoculated in YPD medium and then inoculated into the fermenter until the D600 of the culture reached about 5.2. Using fed-batch fermentation, the high density fermentation of genetically engineered Pichia pastoris was processed in a 5 L bioreaetor. After in- duction for 84 h, the target protein was purified by 6 X His-Tagged Protein Purification Kit. mChIL-18 liposomes and Newcastle disease vaccine were inoculated to chickens. The mChIL-18 was expressed in 5 L fermenter with batch feed methanol and the concentration reached 560 mg/L. The optimum expression conditions as follows：temperature 28℃ ,pH 5.5 and the DO 20 %-30%. After the purification,its purity could be more than 70%. The results showed that 0.2 mL mChIL-18 liposomes could significantly enhance the immunity with a significant increase in the percent age of HI antibody,CD4＋ and CD8 ＋ . The above result lays a foundation for clinical applications of mChIL-18.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2012年第8期1116-1120,共5页
Chinese Journal of Veterinary Science
基金
国家"十一五"科技支撑计划重大资助项目(2012BAK17B05)
国家肉鸡产业技术体系建设资助项目
山东省科技支撑计划资助项目(2007GG20009001)
