EGFP和ALR融合基因表达载体构建及其在HeLa细胞中的表达

从人血液中提取总DNA，利用PCR技术扩增人肝细胞再生增生因子基因，将其插入表达载体pEGFP—C1的多克隆位点中，构建增强绿色荧光蛋白（enhanced green fluorescence protein gene，EGFP）和人肝细胞再生增强因子（human augmenter of liver regeneration gene，ALR）融合基因表达载体pEGFP／ALR，并将其转染Hela细胞系，用含G418的DMEM／F12培养液筛选转基因细胞，然后利用PCR和聚丙烯酰胺凝胶电泳检测转基因细胞中ALR基因的存在及其表达，用荧光显微镜检测EGFP基因的表达．结果显示：得到了构建正确的EGFP和ALR融合基因表达载体；在转基因细胞中，PCR扩增得到1．7Kb的ALR条带，蛋白电泳得到57KD大小条带．与ALR和EGFP融合蛋白大小相符；荧光显微镜下观察到发绿色荧光的Hela细胞．在转基因细胞中，EGFP和ALR同时存在并表达，绿色荧光蛋白可作为报告基因指示目的基因的表达，从而简化了目的基因繁琐的检测手段．     

Cloning and roles of goldfish maternal factor β-Catenin cDNA in embryonic development

Interaction between nucleus and cytoplasm has been focused in the field of animal embryonic development, in which study of maternal factors is required positively. β-Catenin, an important maternal factor in early embryogenesis, has been analyzed in its expression pattern and functions in this paper. We have cloned goldfish β-Catenin cDNA gene and compared it with zebrafish β-Catenin cDNA. High homology was found in cDNA and in amino acid sequences between them, 93% (2227/2384 bp) and 98.5% (768/780 aa) respectively. The expression pattern of β-Catenin by in situ hybridization and the roles of β-Catenin on embryonic development by co-injection of anti-sense RNA and reporter gene, EGFP have been investigated in the whole process of goldfish embryonic development. The results suggest that β-Catenin presents dynamic distribution, mainly locates at body axis, dorsal tissues, head and tail structures after being fertilized. The loss of β-Catenin activity would cause serious destruction of embryo in dorsal tissues and in anteroposterior axes, and leads embryos to die before larva get hatched.     

pBK-CMV⊿-EGFP-SNAT2质粒载体构建及其表达鉴定

SNAT2是SLC38基因家族编码的Na＋偶联中性氨基酸转运蛋白中属于systemA的成员之一．它在谷氨酸一谷氨酰胺循环、肝脏糖质新生等生物通路中发挥重要作用．增强型绿色荧光蛋白EGFP的连接对于SNAT2在细胞表达中的定位以及检测都有很大的帮助．采用PCR扩增和酶切技术将EGFP连接到质粒pBK-CMV△-SNAT2中SNAT2的N端．通过酶切后琼脂糖凝胶电泳和测序验证得到pBK-CMV△-EGFP—SNAT2质粒载体,将构建好的质粒瞬时转染进人胚肾细胞（HEK293cells）用Westernblot和激光共聚焦电子显微镜检测EGFP-SNAT2的表达与亚细胞定位．结果表明：EGFP．SNAT2在细胞中正确表达并定位于细胞膜上．pBK．CMV么．EGFP．SNAT2质粒载体的构建对于进一步研究SNAT2的结构和功能具有重要意义．     

烟草新驱动蛋白NtTKR过表达对酵母生长的影响

为了解烟草新驱动蛋白NtTKR功能信息,构建了EGFP融合的NtTKR基因酵母表达载体并转入酿酒酵母W303诱导表达,荧光显微镜观察结果表明,诱导条件下的转化细胞发出明亮的绿色荧光,表明转入的NtTKR得到了高效表达.转化株在诱导和非诱导条件下培养3d,可见NtTKR过表达菌落比对照小的多;但通过绘制8h内生长曲线发现二者细胞数目无显著差别;表明NtTKR过表达对酵母细胞生长的抑制可能更多的是因为抑制其体积增大,而非分裂速度减慢引起的细胞数目降低.已知NtTKR在烟草受精卵、发育各个时期的胚、幼叶、根尖及茎尖等细胞分裂旺盛部位均优势表达,推测该基因在烟草中可能与上述各部位新生细胞的体积增大相关.     

慢病毒介导的外源基因在人牙髓干细胞中的表达

牙髓干细胞（dental pulp stem cells,hDPSC）是牙源性的间充质干细胞,具有多向分化潜能.已有的研究表明,一些蛋白因子能够诱导牙髓干细胞的牙向分化,但尚无通过过量表达某些关键基因来诱导牙髓干细胞牙向分化的报道.利用绿色荧光蛋白作为报告基因,探究慢病毒介导的外源基因在牙髓干细胞中的表达,分离并鉴定人的恒牙牙髓干细胞,用携带绿色荧光蛋白标记的慢病毒原液感染DPSC,感染病毒后的DPSC进行传代以验证外源基因的稳定表达,并将感染慢病毒后的DPSC与羟基磷灰石/磷酸三钙（hydroxyapatite/tricalcium phosphate,HA/TCP）混合移植到小鼠肾囊膜下培养8周.结果表明：用绿色荧光蛋白标记的慢病毒能够整合到牙髓干细胞的基因组中并获得稳定的表达,感染慢病毒前后的牙髓干细胞增殖率没有发生改变,感染病毒的hDPSC与HA/TCP混合移植到肾囊膜下仍能够产生牙本质牙髓样结构,因此利用慢病毒载体介导的绿色荧光蛋白并不影响牙髓干细胞的生物学特性,说明在进一步利用慢病毒载体研究过量表达基因在牙髓干细胞牙向分化的作用中,绿色荧光蛋白可以作为标记蛋白.     

Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, slw3 and slw6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after activation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.     

增强型绿色荧光蛋白表达对鼠C6细胞生物学特征影响的研究

脂质体包裹质粒pEGFP-N1转染C6细胞,经G418筛选,得到稳定表达绿色荧光的C6-EGFP细胞.细胞计数,MTT,流式细胞术分别测定C6细胞在转染绿色荧光蛋白后增殖性,细胞活力以及细胞周期等生物学特性.C6细胞在转染了绿色荧光蛋白基因后细胞增殖性降低,细胞活力降低,细胞周期也发生了一些改变.     

含有肌球蛋白重链启动子的p-EGFP1质粒的构建

从前列腺组织中提取总DNA，以其为模板经PCR扩增得到肌球蛋白启动子，将扩增片段和处理好的p—EGFP1载体相连，连接产物转入到大肠杆菌HB101中，经筛选得到连有myosin启动子的p—EGFP1重组质粒．用酶切和PCR的方法对重组质粒进行鉴定．成功构建的质粒可用于前列腺平滑肌细胞和成纤维细胞的分离，这将对于阐明前列腺基质增生的机理起到重要作用．     

乳腺癌特异性多肽介导生物大分子体内外效应

探讨乳腺癌特异性多肽PI携带外源性生物大分子靶向抗肿瘤的作用.将分离纯化获得的融合蛋白PI-EGFP与靶细胞MDA-MB-231体外共培养,探讨融合蛋白与靶细胞的结合能力;将此融合蛋白经尾静脉及肿瘤局部注射入荷瘤裸鼠体内,探讨其在肿瘤部位的聚集程度;利用分子克隆技术构建重组原核表达载体pET-28a(+)-pI-tk,诱导表达、分离纯化、鉴定获得的融合蛋白PI-HSV-TK,将不同浓度的融合蛋白与MDA-MB-231细胞共培养,经更昔洛韦(ganciclovir,GCV)作用后,探讨PI-HSV-tk对细胞的靶向杀伤效应.荧光显微镜下观察,在靶细胞内可检测到绿色荧光信号,经尾静脉及局部注射融合蛋白PI-EGFP后,在不同组织器官可见不同强度的荧光信号,在尾静脉注射组的肾脏和肿瘤部位可检测到荧光信号,而局部注射组仅在肿瘤部位可检测到;成功构建了重组原核表达载体pET-28a(+)-pI-tk;分离纯化获得高效表达的PI-TK融合蛋白,SDS-PAGE电泳及Western blotting鉴定融合蛋白的表达正确;CCK-8法及流式细胞仪检测显示,GCV对转导融合蛋白的MDA-MB-231细胞有杀伤作用,IC50值为152.64μg/mL.乳腺癌特异性转导多肽能携带生物大分子进入靶细胞,并携带具有杀伤效应的物质,使其发挥靶向治疗作用,为进一步探讨该多肽作为靶向性载体奠定实验基础和理论依据.