重组人ω干扰素在毕赤酵母中的表达纯化与分析.docx

重组人ω干扰素在毕赤酵母中的表达纯化与分析
摘要
干扰素（Interferon，IFN）是一类具有抗病毒、免疫调节和抗肿瘤等多种生物活性的细胞因子，也是重要的抗病毒免疫药物。本研究旨在通过重组毕赤酵母表达人ω干扰素，进行其纯化与分析。首先使用PCR将人ω干扰素基因克隆到表达载体pPIC9K中，随后通过多步酵母转化，将重组载体引入毕赤酵母中，成功表达了重组人ω干扰素的可溶性表达。经过Ni-NTA亲和层析、DEAE-SepharoseFastFlow离子交换层析和Superdex200gelfiltration层析三步纯化，得到了目标蛋白的高纯度。Westernblot和SDS-PAGE结果表明，纯化后的重组人ω干扰素具有较好的单一性。进一步的质谱分析表明，纯化后的重组人ω干扰素质量为1.92kDa，与理论质量十分接近。本研究为重组干扰素的生产提供了重要参考。
关键词：重组人ω干扰素，毕赤酵母表达，纯化，分析
Abstract
Interferon(IFN)isatypeofcytokinewithvariousbiologicalactivities,suchasanti-virus,immuneregulationandanti-tumor,anditisalsoanimportantantiviralimmunomodulatorydrug.TheaimofthisstudywastoexpressrecombinanthumanomegainterferoninPichiapastorisandpurifyandanalyzeit.Firstly,weclonedthehumanomegainterferongeneintotheexpressionvectorpPIC9KbyPCR.Then,throughmultipleyeasttransformations,therecombinantvectorwasintroducedintoPichiapastorisandthesolubleexpressionoftherecombinanthumanomegainterferonwasachieved.Afterthreestepsofpurification,includingNi-NTAaffinitychromatography,DEAE-SepharoseFastFlowionexchangechromatographyandSuperdex200gelfiltrationchromatography,thetargetproteinwasobtainedwithhighpurity.TheresultsofWesternblotandSDS-PAGEshowedthatthepurifiedrecombinanthumanomegainterferonhadgoodsingularity.Furthermassspectrometryanalysisshowedthatthemassofthepurifiedrecombinanthumanomegainterferonwas19.2kDa,whichwasveryclosetothetheoreticalvalue.Thisstudyprovidesanimportantreferencefortheproductionofrecombinantinterferons.
Keywords:Recombinanthumanomegainterferon,Pichiapastorisexpression,purification,analysis
Introduction
Interferon(IFN)isanimportantcytokinewithvariousbiologicalactivitiessuchasanti-virus,immuneregulationandanti-tumoreffects.Theyhavebeenwidelyusedasantiviraldrugs,immunomodulatorydrugs,andanti-tumordrugsinclinicalpractice.Interferonscanbeproducedbynaturalsources,suchasleukocytesandfibroblasts,orbyrecombinantDNAtechnology.Amongthem,recombinantinterferonshavesignificantadvantages,includinghighpurity,controllableconcentration,andrelativelylowprice,whichmak