【作者】 孙丽；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 硕士

【摘要】 乳铁蛋白可以抑制和杀死许多革兰氏阳性、阴性细菌及一些真菌,具有抗氧化、调节人体免疫反应等重要的生物学功能,可以作为高效补铁剂、防腐剂、抗氧化剂、抗肿瘤剂、潜在的饲用抗生素代替品等,具有广阔的应用前景。CD14是机体免疫反应过程中的关键分子,包括脂多糖(LPS)、肽聚糖(PGN)、磷壁酸(LTA)在内的多种细菌产物都通过CD14的途径激活单核细胞、巨噬细胞、内皮细胞等免疫细胞,释放细胞因子及其它炎症介质,介导一系列生理和病理反应。CD14分子以两种形式存在:膜结合型CD14(mCD14)和可溶性CD14(sCD14)。sCD14可以预防感染、提高机体免疫力并且对一些疾病具有一定的治疗作用。开发和研究乳铁蛋白和CD14已经引起了广泛关注。但目前乳铁蛋白工业化生产成本高,产量不理想,CD14在乳汁等中含量低,又存在其他大量存在的蛋白的影响,大量纯化也很难。所以,近年来注目的焦点是利用基因工程等技术来开发低成本的乳铁蛋白和CD14,期望这两种蛋白得到充分利用。植物作为生物反应器来生产重组蛋白已有许多成功的实例。在现存的各种重组蛋白表达系统中,植物生物反应器是最经济的一种表达系统。利用植物生物反应器生产外源蛋白具有一定的优势,它所表达的蛋白在折叠和组装上和动物细胞的更为相似,从而具有和高等动物细胞表达的产物基本一致的免疫原性和生物活性,并且不会存在被一些动物病毒、病原体污染的可能。但是,植物表达系统还处于发展阶段,存在着外源蛋白表达量低等问题。本工作拟为利用玉米作为生物反应器生产人乳铁蛋白和CD14的研究奠定基础。首先从人乳腺cDNA中克隆得到了乳铁蛋白和CD14的全长cDNA序列,为了提高重组蛋白在转基因植物中的积累量,又从玉米种子cDNA中克隆得到了玉米醇溶蛋白信号肽编码序列。然后,利用基因重组技术用玉米醇溶蛋白信号肽代替了乳铁蛋白和CD14基因原有的信号肽,构建融合基因和乳铁蛋白基因、CD14基因的植物表达载体,采用农杆菌介导法将乳铁蛋白基因、CD14基因转入烟草和玉米,获得了转基因植株。通过对转基因烟草植株进行Western Blot检测,肯定了乳铁蛋白和CD14在烟草中被成功表达。ELISA定量检测结果显示,转基因烟草植株表达的重组蛋白量分别为0.41%(转带有玉米醇溶蛋白信号肽乳铁蛋白基因)、0.46%(转带有玉米醇溶蛋白信号肽CD14基因)、0.17%(转带原有信号肽乳铁蛋白基因)和0.21%(转带原有信号肽CD14基因),更换有玉米醇溶蛋白信号肽序列的融合基因在转基因烟草植株中表达的重组蛋白量成倍高于带原有信号肽序列的乳铁蛋白基因和CD14基因的表达量,证明了玉米醇溶蛋白信号肽的引入确实增加了重组蛋白在转基因植株中的积累量。重组乳铁蛋白抑菌试验结果表明,更换有玉米醇溶蛋白信号肽和带原有信号肽的转基因烟草植株表达的重组乳铁蛋白具有相同的生物活性,说明玉米醇溶蛋白信号肽序列的引入不影响重组蛋白的生物活性。转基因烟草中的表达研究为这两个基因转入玉米中的研究提供了资料。在转基因玉米中,为了进一步提高重组蛋白的积累量,采用了玉米种子胚乳特异性启动子启动乳铁蛋白基因和CD14基因表达,拟将重组蛋白定位在玉米种子胚乳中。转基因玉米检测的工作正在进行中。更多还原

【Abstract】 Human lactoferrin has some important physiological functions such as antimicrobial activities, antioxidant activity and immunomodulatory activity. To date, it has become evident that lactoferrin exerts various beneficial effects against diseases and is safe for health. This enlarges the application potential of lactoferrin. Human CD14 is involved in the innate immunity system and the inflammation reaction. As the receptor for the LPS of gram-negative bacteria or the peptidoglycan of gram-positive bacteria, CD14 elicits a series of physiological and pathological responses. Human CD14 exists either as a membrane-bound protein (mCD14) or as a soluble protein (sCD14). During the past twenty years, the ability of sCD14 to protect against infection and the potential of therapeutic had been described in numerous in vitro and in vivo studies. The research on lactoferrin and CD14 has been arousing more and more interest from the public. However, producing human lactoferrin on a factory scale has a high cost and a dissatisfactory yield. Low concentration of CD14 in milk and the presence of numerous other proteins make it a difficult task to purify a relatively large quantity of CD14. Therefore, to make best use of lactoferrin and CD14, more attention has been focused on using gene-engineering technology to produce low cost proteins.Plants are now considered as viable and competitive expression systems for the production of recombinant proteins. Some proteins have been successfully produced in plants. Compared to other organisms, plants offer several advantages for producing foreign proteins. They have eukaryotic post-translational modification machinery and the correct folding and assembling of protein in plants are very similar to the situation in mammalian cells. Furthermore, plant expression systems present no risk of product contamination by mammalian viruses, pathogens and oncogens, and present a potential for lower cost of production when using field crops. However, plant expression systems are developing, and there are some problems to solve such as low expression. The study was on a mission to lay a foundation for using maize to produce human lactoferrin and CD14. To increase the expression of recombinant proteins, the signal peptide from maize zein was used. We had engineered two constructs containing either the native signal peptide from human lactoferrin/CD14 or signal peptide from maize zein fused to human lactoferrin/CD14 encoding cDNA. The plant expression vectors contained human lactoferrin and CD14 gene were introduced into tobacco and maize by Agrobacterium-mediated. Western blot analysis showed human lactoferrin and CD14 had expressed in transgenic tobacco under the control of the CaMV-35S promoter. The concentration of recombinant protein in tobacco was assessed by ELISA. The result suggested that the use of signal peptide from maize zein increased the expression level of recombinant protein. The recombinant human lactoferrin expressed in tobacco had antibacterial ability for both constructs.The study in transgenic tobacco provided some important data for the expression study of human lactoferrin and CD14 in maize. Transgenic maize plants with expressing the two proteins under the maize seed endosperm specific promoter for increasing the expression level of recombinant proteins in endosperm of seed were just obtained, and the analyses of these maize plants are carring out.更多还原