猫干扰素omega基因的可溶性表达及重组蛋白纯化方法和活性研析.pdf

摘要 胞因子。为了研制我国猫干扰素生物制剂及干扰素佐剂，并为研究FelFN．∞的蛋白结构奠定基础， 型FelFNo进行了亚克隆。扩增的目标PCR产物纯化回收后，经BamHI和HindIII双酶切，插 HI和H／rid (Amp)的LB平板，筛选阳性克隆，并对重组子进行了Bam 亲和层析纯化rFeIFN．∞，再经SDS电泳和WesternBlot检测，分析纯化的重组蛋白。并 且在猫肾上皮细胞(CRFK)一水泡性口炎病毒(VSV)系统上，采用细胞病变抑制法测定rFelFN．∞ 的抗病毒活性。 预测大小一致。SDS．PAGE检测和Western 白的比活均可达到10 7IU／mg。且这五个亚型的猫干扰素。蛋白的活性并不存在特别显著的差异。 本研究的完成为生物工程发酵生产猫重组干扰素打下了坚实的基础。 关键词：猫。型干扰素，可溶性表达，纯化，抗病毒活性 Abstract that viral Felineinterferonsa sharethe toinhibit representfamilyofcytokines capacity andexerteffectsonimmunefuncdon．InordertO and IFN to developbiologicaladjuvantagent，and establishthefoundationfortheresearchofthe ofuniversal proteinstructure，apair primers BamHIandHindIIIrestrictionsitewere tothefiveFeIFN-COthatwe designed．according gene have cloned subclonethefivenew FeIFN-∞．After and PCR before，to subtype purification were withBamHIand砌d clonedintotheBamHI Ill productsdigested HI．thell and脚”d vector whichcontainsT7 intoE．coliTOPlOF’ expressionpET-His promoter．TransformpET-His cellandscreen clonesafterthebacterialwerecultured onLB competent positive ovcrnightplates recombinant supplementedwi山ampicillin．Correct and wereobtainedafter FeIFN—∞2、pET-His／FeIFN一0)3、pET-His／FeIFN一0)4pET-His／FeIFN-(05 transformantsweleidentifiedBamHIandH／ndIll and