牛α0干扰素在毕赤酵母中的分泌表达及抗病毒活性鉴定  

Expression of the Bovine Interferon Alpha0 with Antiviral Activity in Pichia Pastoris

作　　者：彭统全 邵建伟[1] 张润祥[1] 曹翀[1] 马波[1] 高明春[1] 王君伟[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030

出　　处：《畜牧兽医学报》2015年第1期124-129,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：现代农业(奶牛)产业技术体系(CARS-37);国家科技支撑计划项目(2012BAD12B05);国家科技支撑计划子课题(2012BAD12B03-3);黑龙江省科技攻关项目(GA09B302)

摘　　要：克隆牛α0干扰素(BoIFN-α0)成熟肽基因,使其在真核细胞中表达,并研究其表达蛋白质的活性。通过PCR方法扩增得到BoIFN-α0成熟肽基因,然后将其连接到含分泌信号肽序列的Pichia pastoris表达载体pPICZαA上,构建重组质粒pPICZαA-BoIFN-α0,重组质粒经PmeⅠ酶切线性化后电转化宿主菌GS115。转化子经100μg·mL-1 zeocinTM筛选和PCR鉴定,阳性重组菌经甲醇诱导后实现了BoIFN-α0在毕赤酵母系统中的分泌表达。结果表明:SDS-PAGE和Western blot结果显示表达产物相对分子质量约为18和21ku,推测表达产物发生了一定程度的糖基化。细胞病变抑制试验结果表明,重组BoIFN-α0具有较高的抗病毒活性,达到5.72×106 U·mg-1。这些研究结果为牛IFN-α的更深层次应用奠定了基础。In this study,we cloned BoIFN-α0mature peptide gene and expressed it in the eukaryocyte,and also studied the activity of this protein.To achieve secretary expression of bovine interferon-α0(BoIFN-α0)in Pichia pastoris,the BoIFN-α0mature peptide gene was synthesized by PCR and inserted into the secreted expression vector pPICZαA.The Pme Ⅰ linearized resultant recombinant plasmid was transformed into P.pastoris GS115 by electroporation and the positive recombinant strains P.pastoris were selected by 100μg·mL-1 of zeocinTMand identified by PCR with the AOX1 primers and the specific primers.The positive recombinant strains were induced with methanol and achieved secretory expression in P.pastoris Yeast expression system.The products in culture medium were detected by SDS-PAGE and Western blot.The SDS-PAGE results showed that the recombinant protein was expressed in the supernatant with molecular of 18 and 21kDa(with the difference of glycosylation).Furthermore,the BoIFN-α0was able to efficiently inhibiting virus replication in vesicular stomatitis virus(VSV)/MDBK cells detection system and its antiviral activity was about 5.72×106 U·mg-1.These results provided a basis for further application of BoIFN-α.

关 键 词：牛BoIFN-α0亚型 巴斯德毕赤酵母 分泌表达 抗病毒活性 

分 类 号：Q78[生物学—分子生物学]