猪β-干扰素的基因改造表达及其抗猪繁殖与呼吸综合征病毒活性测定  

Gene Modification and Expression of Porcine Interferon Beta and Its Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

作　　者：张显浩[1] 苏丹萍[1] 张艳萍[1] 蒋爱翔[1] 贺东生[1] 

机构地区：[1]华南农业大学兽医学院广东省人畜共患病重点实验室,广东广州510642

出　　处：《中国畜牧兽医》2011年第10期63-67,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31072138);番禺科技局(2009-z-23-4)资助

摘　　要：本研究根据GenBank上登录的猪β-干扰素基因成熟肽核苷酸序列(mPoIFNβ),在保持原猪β-干扰素蛋白序列不变的基础上,对猪β-干扰素基因进行了毕赤酵母偏嗜性改造,并构建了毕赤酵母重组表达质粒pPICZαC-PoIFNβ。pPICZαC-PoIFNβ经SacⅠ酶切线性化后,电击转化导入感受态的毕赤酵母菌株X-33中,转化子经YPDS+Zeocin抗性平板筛选和PCR鉴定后获得多株阳性菌株。阳性酵母菌株经甲醇诱导分泌表达了重组PoIFNβ,其表达量约为127.9mg/L。表达产物经SDS-PAGE和Western blotting检测,结果表明,表达产物为分子质量约25和28ku的混合物,并且二者都可与PoIFNβ阳性血清结合。以细胞病变抑制法测定重组β-干扰素在BHK-21细胞上的抗水泡性口炎病毒活性为2.8×103 IU/mL;对猪繁殖与呼吸综合征病毒(PRRSV)在Marc-145细胞上抗病毒活性达到1.6×103 IU/mL。Based on the sequences of porcine interferon-β gene and the mature gene（mPoIFNβ） available in GenBank,the gene encoding porcine interferon-β was modified for adapting codons of Pichia pastori and a recombinant vector pPICZαC-PoIFNβ was developed,while maintaining the same protein sequence of porcine interferon-β.The plasmid pPICZαC-PoIFNβ was linearized with SacⅠ and electroporated into Pichia pastoris X-33.The recombinant colonies were selected in high resistance culture of YPDS＋Zeocin and identified by PCR.Induced by menthanol,a few positive clones were obtained secretory highly expressed the PoIFNβ protein with a protein concentration of 127.9 mg/L.The expressed supernatant was identified by SDS-PAGE and Western blotting.On the gel and membrane there were two major protein bands with molecular weight about 25 and 28 ku and they showed positive reaction with anti-PoIFNβ antibody.The CPE suppression by PoIFNβ was tested for vesicular stomatitis virus（VSV） in BHK-21 cell line and the porcine reproductive and respiratory syndrome virus（PRRSV） in Marc-145 cell line.The antiviral activity of the expressed PoIFNβ on BHK-21 cell line challenged with VSV was 2.8×103 IU/mL,and 1.6×103 IU/mL on the Marc-145 cell line challenged with PRRSV.

关 键 词：猪β-干扰素 毕赤酵母 分泌表达 抗病毒活性 猪繁殖与呼吸综合征病毒 

分 类 号：Q78[生物学—分子生物学]