【作者】 曹海军；

【导师】 姚火春； 樊生超；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 乳铁蛋白(Lactoferrin,简称LF)是一种多功能的糖蛋白,主要存在于哺乳动物的乳汁、眼泪等外分泌物中,乳汁尤其是初乳中LF的含量最高。LF不仅具有广谱的抗菌和抗病毒的作用,而且还能够增强机体抗感染、抗肿瘤和免疫能力。通过30%饱和硫酸铵沉淀、50%饱和硫酸铵沉淀、85%饱和硫酸铵沉淀——三步沉淀法和一步肝素琼脂糖亲和柱亲和层析进行天然猪乳铁蛋白的分离纯化,SDS-PAGE分析表明,获得的猪乳铁蛋白的相对分子量为80kDa。分别用制备的天然猪乳铁蛋白免疫鸡和兔子,制备高免血清,建立了测定猪乳铁蛋白的双夹心酶联免疫法(ELISA),并将该法运用于分离纯化天然猪乳铁蛋白各步骤,计算出了猪乳铁蛋白最终得率与纯度:65.6%,91%。Pichia pastoris表达系统是近年来迅速发展起来的一种真核表达系统。Pichiapastoris是一种甲醇酵母,可以在以甲醇为唯一碳源和能源的培养基中生长。在表达外源蛋白时,不仅具有原核生物生长快、操作简便、成本低的特点,而且具备哺乳动物的翻译后加工修饰的功能,从而表达出有生物活性的外源蛋白。应用RT-PCR方法获得小型猪乳铁蛋白(pLF)基因,将其插入到pMD19-T simple载体中,构建克隆载体pMD-pLF。克隆的pLF基因片段全长2102bp,与GenBank上猪乳铁蛋白序列(登录号:L77887)的同源性达99%以上。对克隆载体pMD-pLF进行双酶切,将得到的2102bp长的pLF基因片段以正确的阅读框架定向克隆入含乙醇氧化酶启动子和分泌信号肽序列的Pichia pastoris表达载体pPICZαA中。通过电击法将经SacI酶切线性化的pPICZαA-pLF转化到毕赤酵母菌株GS115中,经Zeocin抗性浓度梯度筛选获得高抗性重组菌株,0.5%甲醇诱导表达。表达产物经TCA/DOC浓缩后进行SDS-PAGE和Western-blot鉴定。结果显示,分泌于胞外的重组蛋白的分子量约为80kDa,与天然猪乳铁蛋白的分子量基本一致。Western-blot结果表明,表达产物可与猪乳铁蛋白抗体发生特异性反应。上述结果说明本试验成功实现了pLF在毕赤酵母系统中的分泌表达。更多还原

【Abstract】 The lactoferrin as an iron-binding glycoprotein, is presented in numerous exocrine secretions of mammals, including milk, tears, saliva, et al.Milk,especially porcine colostrums contains lactoferrin at a much higher level than others. Lactoferrin is a protein with multi-functions,including broad spectrum antibacterial properties, antiviral activity, anticancer activity and immunoregulatory activity.The methylotrophic yeast Pichia pastoris has been developed as an efficient expression system for foreign proteins. It can grow on methanol as a sole carbon and energy source. As a eukaryotic expression system, Pichia pastoris has many advantages over prokaryotes and other higher eukaryotes. It is as easy to manipulate and culture as prokaryotes and is able to process posttranslational modifications as other higher eukaryotes.In this experiment,LF was separated and purified by 3 times salt-out and 1 time gel chromatography from porcine colostrums, and measured by SDS-PAGE. For the acquired lactoferrin, the molecular weight was estimated at 80kDa , and the purity was about 91%. Antibody against LF was achieved by injection of rabbits and chicken with LF.Lactoferrin gene of Chinese miniature pig was amplified using RT-PCR, the PCR products were inserted into pMD19-T vector routinely, and the positive recombinants were identified by endonuclease digestion,PCR and DNA sequencing. Sequence analysis found 99% homology with other sequences in GenBank. Digested with XhoI and XbaI, a fragment 2102bp of the PCR product was cloned into Pichia pastoris expression vector pPICZαA containing the inducible promoter of the alcohol oxidase-1 gene(AOX1) and theα-mating factor signal peptide. The recombinant plasmid of pPICZαA-pLF was lineamized by Sad and transformed into GS115 by electroporation. The mufti-copy insertion transformants were screened by Zeocin-resistance and induced by 0.5% methanol. The Pichia pastoris culture medium was concentrated by TCA/DOC and characterized using SDS-PAGE and Western blot. The results showed that the rPLF with a molecular weight of approximately 80kDa which was similar with nature pLF was well expressed in Pichia pastoris.更多还原