摘要
sag基因家族是制备基因工程疫苗重要的候选基因。本研究通过在毕赤酵母Pichiapastoris中表达柔嫩艾美耳球虫Eimeriatenella表面抗原基因sag10,来探讨sag10表达后的生物学活性。本文首先提取柔嫩艾美耳球虫北京株第2代裂殖子总RNA,根据GenBank报道序列(AJ586552)设计引物,应用RT-PCR技术扩增得到鸡球虫表面抗原sag10基因。然后将sag10与真核表达载体pPIC9K连接,构建了pDQ052分泌型真核表达质粒,并转化毕赤酵母GS115,利用G418抗性筛选多拷贝重组菌株,进行优化表达。SDS-PAGE和Westernblot检测表达结果,在43kDa处有明显的免疫印迹条带,表明目的蛋白得到表达,而且能被特异性抗体所识别,说明表达的SAG10蛋白具有生物学活性。
Total RNA was extracted from the Beijing strain of Eimeria tenella and used as the template for the cloning of the sag10 gene by RT-PCR with specific primers designed according to the reported sequence in GenBank. The expression plasmid pDQ052 was constructed and transformed into Pichia pastoris GS115. Multi-copy Pichia pastoris strains were screened by G418. The selected strain was cultured in 100 mL flasks and the supernatant was analyzed by SDS-PAGE and Western blot. The results indicated that the SAG10 protein was successfully expressed and secreted in a soluble form with a molecular weight of approximate 43 kDa in recombinant Pichia pastoris when induced by 0.5 % methanol.
出处
《寄生虫与医学昆虫学报》
CAS
2006年第4期199-203,共5页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家"863"计划课题(2003AA241160)
中国-丹麦政府间国际合作项目
