柔嫩艾美耳球虫钙依赖蛋白激酶3基因在毕赤酵母中的表达及分析

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 tenella calcium-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1-4d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.

Analysis and Expression of Eimeria tenella Calcium-dependent Protein Kinase-3 Gene in Pichia pastoris

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.