摘要
旨在真核表达系统中高效表达柔嫩艾美耳球虫钙依赖蛋白激酶3(Eimeria tenellacalcium-dependent protein kinase-3,EtCDPK3),获得有活性的天然蛋白,利用毕赤酵母表达系统对该基因进行了表达。将EtCDPK3基因连接到毕赤酵母表达载体pPIC9K上,构建重组质粒pPIC9K-EtCDPK3。重组质粒通过电击转化入酵母细胞GS115后,用组氨酸缺陷培养基和G418分别进行筛选,获得含重组质粒的酵母表达细胞。重组酵母细胞在含1%甲醇的BMMY培养基中诱导产生目的蛋白,培养收集1-4 d的部分上清。经SDS-PAGE检测,所表达的蛋白相对分子质量约为49 kD。Western blotting表明,该蛋白能与兔抗EtCDPK3血清特异性结合。结果表明,柔嫩艾美耳球虫CDPK3基因在毕赤酵母中成功地进行了表达。
In order to efficiently express calcium-dependent protein kinase-3（EtCDPK3） of Eimeria tenella in eukaryotic expression system and obtain the active and natural protein,EtCDPK3 gene were ligated to Pichia pastoris expression vector pPIC9K.The recombinant plasmid pPIC9K-EtCDPK3 was constructed successfully.After the recombinant plasmids were transformed into yeast cells GS115 by electroporation technology,the positive yeast cells containing recombinant expression plasmid which were screened with histidine-deficient medium and G418 were obtained.The protein was produced by recombinant yeast cells which were induced by 1% methanol in BMMY medium.Part of supernatant were collected from 1 d to 4 d.Results showed the target protein band of about 49 kD was identified by SDS-PAGE.Western blotting analysis demonstrated that the target protein could bind specifically to rabbit serum of anti-EtCDPK3.These results showed that Eimeria tenella CDPK3 gene had been successfully expressed in Pichia pastoris,which have provided the foundation for further studying function of the protein.
出处
《生物技术通报》
CAS
CSCD
北大核心
2012年第4期108-112,共5页
Biotechnology Bulletin
基金
上海市自然科学基金项目(09ZR1438700)
中央级公益性科研院所基本科研业务费项目(2011JB01)
