【作者】 葛丽；

【导师】 尹逊河； 李震；

【作者基本信息】 山东农业大学 ， 基础兽医学， 2003， 硕士

【摘要】 干扰素（IFN）是一类重要的细胞因子，具种属特异性，在同种细胞上具有广谱的抗病毒、抗细胞增殖、免疫调节等多种生物活性，研究表明，利用干扰素防治家畜的病毒性疾病是一种理想的治疗方式。但是，动物体内一般情况下不产生干扰素，国内外学者曾尝试利用病毒感染诱导动物机体白细胞产生干扰素，结果仅产生极其微量的IFN且生产成本高，难于提取纯化。随着DNA重组技术的发展，国内外亦有利用原核表达系统表达重组IFN蛋白的报道，但表达的目的蛋白常以包涵体形式存在，提取纯化操作步骤复杂、蛋白活性不高。鉴于以上研究现状，本研究首次尝试利用真核表达系统表达猪α干扰素重组蛋白。 近年来，甲醇营养型酵母表达系统的研究得到迅速发展，因其具有高表达、高稳定、高分泌、能对表达的外源蛋白进行正确的折叠、加工，利于下游分离纯化操作、能大规模发酵生产等诸多优点，国内外已经利用此系统表达了多种对人类有益的动植物及微生物活性蛋白，但对猪α型干扰素的酵母表达还未见报导，本研究是国内外首次采用此系统表达猪α型干扰素。 本研究以猪α干扰素重组质粒PGEX-IFN为模板，运用常规PCR方法扩增得到猪α干扰素基因的成熟编码序列。将此基因与毕赤酵母分泌型表达载体pPICZαA连接，构建pPICZαA-IFN重组质粒，并用常规CaCl₂法转化大肠杆菌JM109进行扩增。提取重组质粒，酶切、PCR扩增和DNA序列分析鉴定阳性克隆。阳性重组质粒经纯化处理后用BioRad公司电转仪电转化毕赤酵母菌株KM71H。抗生素Zeocin^TM浓度梯度筛选高拷贝菌株，分别提取这些高拷贝菌株的基因组DNA，以此为模板做PCR反应鉴定阳性克隆。经上述实验过程筛选出的高拷贝重组菌株分别通过BMGY／BMMY培养基和YPG培养基、用甲醇诱导表达，诱导菌液上清 葛丽：猪a干扰素基因在毕赤酵母中的分泌表达通过三氯醋酸沉淀蛋白处理，做 SDS－PAGE和 West。印迹实验，结果表明表达产物为重组猪口干扰素融合蛋白。将筛选出来的重组菌株诱导144小时，取菌液上清液用VSV／WISH系统检测重组蛋白的抗病毒活性为4．l＊I04lU／ffiL o更多还原

【Abstract】 Interferon (IFN), a family of cytokines, have activities on interfering with the replication of various viruses, decreasing cell proliferation and modifying immunological processes. The research about IFN indicates, it is a kind of ideal therapy that IFN act on healthy or unhealthy animals to prevent or cure many kinds of diseases infected by virus. However, animal organisms could not produce IFN commonly, many scholars had attempted to use virus infect animals in order to induce leukocyte to produce IFN. As a result, a very small quantity of IFN was produced and also the cost of production was very high, it was difficult to extract and purify IFN protein. With the development of DNA recombination technology, there are also some reports about the expression of recombined IFN protein by prokaryotic expression system, but the expressedproteins often consist in the form of inclusion body= The operations about extracting and purifying proteins are very complicated, biological activity of proteins are not high. Therefore, this research is the first time to attempt to express porcine IFN-a(PoIFN-a) by eukaryotic expression system.Recently, the expression system of methylotrophic yeast has been developed quickly. As a result of its many advantages, such as high expression, high stability, high secretion and so on, many kinds of Protein, including animal, plant, microorganism proteins, have been expressed by this system in the world, but we have not seen any report about yeast expression of PoIFN-auntil now, this research is the first example in the world that PoIFN-a is expressed by methylotrophic yeast system.This research use recombinant plasmid PGEX-IFN as template, mature PoIFN-a gene was obtained by PCR method. The expression vector pPICZaA with the PoIFN-a gene was constructed by restrict enzyme digestion and ligation, then transformed into E.coli JM109 in order to extract recombinant plasmid from JM109. After DNA sequencing was correct, it was transformed into P.pastoris KM71H strain by electroporation. Highly resistant recombinant was isolated by increasing concentrations of Zeocin? positive clone was selected by PCR .The positive clone was induced with methanol . The results of SDS-PAGE and Western Blot showed that the product was recombinant PoIFN-a fusion protein. Biological activity of this fusion protein was detected by VSV/WISH system, the result was 4.104.1 104IU/m=更多还原