摘要
将猪白细胞介素-2(pIL-2)的完整cDNA序列克隆到甲醇酵母(Pichiapastoris)表达载体pPIC3 5K中,在E.coli体系中鉴定得到阳性克隆子pPIC3 5K-IL2。将pPIC3 5K-IL2经SacI线性化后,转化入经LiC1致敏的P.PastorisGS115菌株中,得到的重组菌株经1%浓度甲醇诱导后,利用SDS-PAGE电泳,斑点杂交及去糖基化酶消化证实,在培养上清中获得了分泌型表达的猪白细胞介素-2(蛋白分子量约为20KD),其表达量可达到80mg/L。用CTLL-2细胞进行活性检测,生物学活性可达2×106IU/ml。对其表达情况进行时间梯度分析,确定第5天表达量达到最高点,为最佳诱导时间;分析连续4个批次的重组菌株表达情况,结果表明重组菌株在适当菌体浓度下连续培养均能稳定、高效的表达外源蛋白pIL-2。本实验为进一步大规模生产pIL-2提供了理论依据。
The intact Porcine Interleukin- 2 (pIL- 2) cDNA sequence was cloned into pPIC3.5K (expression vector of Pichia pastoris) to construct the expression plasmid pPIC3.5K-IL2.The pPIC3.5K-IL2 was further linearized and transformed into P.Pastoris GS115 strain.After being induced by 1% methanol,the recombinant strain excreted a protein in supernatant of culture,with molecular weight of 20 KD approximately,which was proved to be Porcine Interleukin- 2 through SDS-PAGE and Dot Blotting.Its expression level is up to 80 mg/L and bio-activity unit 8×10~6 U/ml.In a 9 day induction experiment,the daily expression status was measured.The results suggest highest level in the fifth day.And we observed the continual expression status of the recombinant strain in 4 test batches and found a stable,high and continual pIL- 2 expression in opportune concentration.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2004年第2期208-212,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家863高技术研究和发展计划项目基金(2001AA213051)
