【作者】 郭学双；

【导师】 薛仁宇；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 绿色荧光蛋白与红色荧光蛋白自发现以来,就因易于检测、荧光稳定、无毒害、种属通用性、易于构建载体、可进行活细胞定位定时观察等特性而得到广泛应用。其应用领域涉及生命科学的各个领域,包括研究基因表达的调控元件、增强子捕获、蛋白定位、研究基因表达的时空特性和空间定位、发育分子机理、筛选药物、临床检验、转基因动物和植物的筛选标记以及对病毒颗粒定位、蛋白质相互作用等方面。因此我们构建了含双荧光报告基因的不同转基因载体分别用于不同目的的转基因实验。首先,为了探索团头鲂β-actin启动子在家蚕BmN细胞和家蚕体内的活性,将转基因载体pigA3GFPAβdsRed和辅助质粒混合,通过脂质体介导转染BmN细胞和利用精子介导法导入家蚕受精卵,48 h后,可以观察到同时发绿色荧光和红色荧光的家蚕细胞,7天后,可观察到蚕卵具有绿色荧光和红色荧光,表明来自团头鲂的β-actin启动子在家蚕BmN细胞中具有活性,随着稳定转化细胞的传代,绿色荧光稳定存在,而红色荧光会逐渐减弱直至检测不到,暗示β-actin启动子不能在家蚕中用于驱动外源基因稳定表达。然后,为探索来源于昆虫的piggyBac(PB)转座子在鲤科鱼转基因中的适用性,在PB转座子基础上构建了两个转基因载体:pigA3GFP-CMVDsRed-IEneo与pigA3GFPAβDsRed,每个转基因载体分别与辅助质粒pigA3、pigCMV组合对草鱼肾脏细胞CIK进行共转染,都得到了同时具有绿色荧光和红色荧光的CIK细胞,说明启动子PAβ、PA3、Pcmv在CIK细胞具有启动活性。根据转化细胞可稳定传代和PCR验证结果,认证外源基因插入到细胞基因组,证明PB转座子在CIK具有转座活性。将辅助质粒pigA3分别与转基因载体pigA3GFP、pigA3GFPAβDsRed以脂质体-精子介导法共同导入金鱼卵细胞,根据绿色荧光和红色荧光的共表达筛选转基因金鱼,再经PCR、Southern杂交鉴定,表明得到了转基因金鱼,说明PB转座子在金鱼体内具有转座活性。此外,为了探索家蚕核型多角体病毒的多角体蛋白包埋特性,构建了含有绿色荧光蛋白基因(gfp)和红色荧光蛋白基因(dsRed)的表达载体pIZT-DsRed。用该载体转染家蚕卵巢细胞系BmN,并以吉欧霉素(zeocin)筛选得到稳定表达绿色荧光蛋白和红色荧光蛋白的细胞系,表明表达载体pIZT-DsRed可以作为转基因载体在BmN细胞使用;进一步以野生型家蚕核型多角体病毒(BmNPV)感染该细胞系,荧光观察发现部分多角体可以发出绿色荧光和红色荧光,Western blot实验结果证实多角体中含有GFP蛋白,显示多角体病毒可以将病毒粒子以外的其他成分包埋进多角体,表明多角体蛋白的包埋机制中存在非特异性识别包埋现象。更多还原

【Abstract】 Since being discovered, green fluorescent protein and red fluorescent protein were used widely in many biological research fields because of their easy detection, fluorescence stablity, non-toxic, species universal, easy construction, real-time location in living cells and so on. Their applications involved in every field of life science, including the research of regulatory elements, enhaner capture, protein localization of gene expression, expression phase, distribution, molecular mechanisms of axiology, screening drugs, clinical testing, labeling of the transgenic animals and plants, localization of virosome, interaction of proteins and so on. Therefore we constructed different transgenic vectors with dual fluorescent reporter genes for different purposes.First, in order to explore the activy of the bluntnose black bream (Megalobrama amblycephala)β-actin promoter (PAβ)in silkworm (Bombyx mori) BmN cells and in vivo, we transferred the transgenic vector pigA3GFPAβDsRed and helper plamid into the BmN cells by the liposome-mediated gene transfer method and into the silkworm eggs by sperm-mediated gene transfer method respectively. 48 hours later, both green fluorescence and red fluorescence were observed in the BmN cells and 7 days later the two kinds of fluorescence were detected in the fertilized eggs of the silkworm. These results suggested that PAβof bluntnose black bream could work in BmN cells. With the steady passage of the transformed cells, the green fluorescence existed stably but the red fluorescence reduced gradually, suggested that the activity of PAβin the BmN cells was weakened because of its susceptible to some cytoline in the BmN cells.Second, to explore the applicability of the transposon piggyBac derived insects, two transgenic vectors pigA3GFPAβDsRed and pigA3GFP-CMVDsRed-IEneo based on transposon piggyBac were constructed. Both of the green fluorescence and the red fluorescence were detected in the transfected Ctenpharyngoden Idellus kidney (CIK) cells with the transgenic vectors and the helper vector pigA3 or pigCMV in which the transpoase gene was driven by cytomegalovirus (CMV) promoter (PCMV). Moreover the transgenic vector pigA3GFP and pigA3GFPAβDsRed were introduced into eggs of the goldfish (Carassius auratus) by sperm-mediated gene transfer. Finally the transgenic pigA3GFP and pigA3GFPAβDsRed goldfishes were obtained and verified by PCR and Southern blotting, among which the one 2-month-old transgenic pigA3GFPAβDsRed goldfish was dissected and both the green fluorescence and the red fluorescence could be detected in several organs, therefore we suggested that the transposon piggyBac was a potentially powerful tool for producing transgenic fish.Third, to explore the profile of immobilization of Bombyx mori nuclear polyhydrosis virus (BmNPV) polyhedron, we firstly constructed both gfp and DsRed contained vector pIZT-DsRed which was used to transfect BmN cells to generate a stable pIZT-DsRed-transformed BmN cell line by screened with zeocin. Then the wild type BmNPV was employed to infect the cell line most of which expressed gfp and DsRed simultaneously so as to observe newly formed polyhedron. The results of emitting green fluorescence and red fluorescence from isolated BmN polyhedron revealed that foreign proteins could be embedded into the polyhedron on a nonspecific recognition way.更多还原