文档介绍：中文摘要为了在我国研制生物工程重组猪干扰素类制剂,防制猪病毒性传染塑和进一步开展猪干扰素的分子生物学研究,本研究率先利用毕赤酵母(Pichia pastoris)。(PolFN-yx)基因序列, 在其成熟肽和终止密码子序列问分别设计了含EcoRl和BamHl酶t:JJ位点的一对引物, 采取聚合酶链式反应(PCR)法对PolFNq'2进行了亚克隆。PCR产物经纯化回115(后以EcoRI和BamHl双酶切,(PHOI) 分泌信号肽之后,转化大肠杆菌JMl09感受态细胞,用含氨苄青霉素(Amp)的LB 平板筛选阳性克隆,并对重组菌进行PCR鉴定、EcoRI和BamHl双酶切鉴定以及序列测定。-T2。-)'2电穿孔导入毕赤酵母GSll5/His’株t以选择性培养基基本甲醇培养基(Minimal Methanol,MM)和基本葡萄糖培养基(Minimal Dextrose,MD)在30"(2培养2天后筛选阳性酵母重组菌(HisV Mots)。为了挑选高效表达株,将筛选出的阳性转化子首先在缓冲甘油复合培养基(Buffered Glycerol- complex Medium,BMGY)(以甘油为碳源)中培养,待其生长至饱和状态,离心去除BMGY,换入诱导培养基一缓冲甲醇复合培养基(Buffered plex Medium,BMMY)(以甲醇作诱导物),在30"C诱导表达5--,6天,再离心收集菌体、超声波裂解,-7的酵母菌株。然后用Western --blot试验分析鉴定。将鉴定后的重组干扰素蛋白充分透析后,采用牛肾细胞(MDBK)一滤泡性口炎病毒(VSV)系统,使用细胞病变抑制法测定rPolFN-y2的抗病毒生物学活性。7/撬尸乎占筋琵/~争(本研究成功的亚克隆了猪的IFN吖基因,PolFN-y2基因全长439bp,编码146个氨基酸组成的成熟多肽。构建的整合表达质粒pHIL—s1/PolFN-T,在酵母重组菌GSll5/ His4中成功表达了蛋白分子量为18kDa左右的IFN糖基化蛋白。抗病毒生物学活性测定的结果表明rPolFN-y2的抗病毒活性的范围为450-540 unit/ml。本研究为生物工程发酵生产猪熏组卜干扰素打下了的基础。)7。关键词猪II型干扰素毕赤酵母基因表达抗病毒活性 ABSTRACT ()is ahomodimoric glycosylated hat plays important roles on ,antipreliferetive,and immonomodulatory order todevelop binant PolFN吖preparations preventing porcine viral infectious disease, and tofurtherexpound theresearch ofcytokines in thefield ofmolecular biology,PolFN吖 was firstlyexpressed inPichia pastoris expression system pair ofprimers containing EcoRl and BamHI restriction sites were designed,according to the cloned PolFN-7 gene in our previous research,then PolFN—T gene was subcloned by PCR-The PCR product was digested withEeoRI and BamHI,and ligated toyeast expression vector pHIL-SI atEcoRI and BamHI binant plamid was transformed into Ecoli petent cellsand positive clones were screened afterthebacterial was cultured overnight on LB plate supplemented with were identified by PC