用Bac—to—Bac杆状病毒表达系统高效表达绿色荧光蛋白标记的HBVe抗原

通过基因工程操作,使乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）基因与绿色荧光蛋白基因（ＧＦＰ）融合,用新型Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达系统在昆虫细胞中高效表达了ＨＢｅＡｇ－ＧＦＰ双功能融合蛋白。经ＥＬＩＳＡ法和荧光显微镜观察证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＢＶ的ｅ抗原活性,为免疫诊断新方法的建立进行了有益的探索。     

用细菌/杆状病毒系统在昆虫细胞中表达GFP与HCV抗原融合蛋白

用细菌／杆状病毒（Ｂａｃ－ｔｏ－Ｂａｃ）系统在昆虫细胞中高效表达了绿色荧光蛋白（ＧＦＰ）与ＨＣＶ抗原的双功能融合蛋白,经ＥＬＩＳＡ测定和荧光显微镜观查证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＣＶ的抗原活性,实现了用绿色荧光蛋白等分子标记抗原,为免疫诊断新方法的建立打下了理论基础．     

绿色荧光蛋白与 HCV 核心蛋白的融合及在大肠杆菌中的高效表达

利用基因工程重组技术获得了绿色荧光蛋白（ｇｆｐ）基因与ＨＣＶ核心蛋白（ｃｏｒｅ）基因的嵌合体,并在大肠杆菌中高效表达了４８ｋＤａ的融合蛋白,经ＤｏｔＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ免疫活性分析证实,融合蛋白仍具有ｃｏｒｅ抗原的三个免疫活性部位,同时用荧光显微镜观察并用荧光光度计测定了大肠杆菌表达的融合蛋白的荧光光谱,结果证实,我们在大肠杆菌中表达的ＧＦＰｃｏｒｅ融合蛋白既能发射易于检测的绿色荧光,又具有ＨＣＶ核心蛋白的抗原活性,实现了用绿色荧光蛋白等分子标记抗原,为免疫诊断新方法的建立,打下了理论基础。     

绿色荧光蛋白与 HCV 核心蛋白的融合及在大肠杆菌中的高效表达

利用基因工程重组技术获得了绿色荧光蛋白（ｇｆｐ）基因与ＨＣＶ核心蛋白基因的嵌合体,并在大肠杆菌中高效表达了４８ｋＤａ的融合蛋白,经Ｄｏｔ－ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ ｂｌｏｔ免疫活性分析证实,融合蛋白仍具有ｃｏｒｅ抗原的三个免疫活性部位,同时用荧光显微镜观察并用荧光光度计测定了大肠直菌表达的融合蛋白的荧光光谱,结果证实,我们在大肠杆菌中表达的ＧＦＰ－ｃｏｒｅ融合蛋白既能发射易于检测的绿色荧光,又具     

粒细胞—巨噬细胞集落刺激因子／白细胞介素—3融合蛋白的表达研究

利用ＰＣＲ扩增得到粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）、白细胞介素－３（ＩＬ－３）完整基因片段,将其分别克隆ｐＧＥＭ－Ｔ构建成ＧＭ－ＣＳＦ／ＩＬ－３融合蛋白基因,ＤＮＡ序列与设计预期一致。将得到的融合蛋白基因克隆对７２ＲＮＡ聚合酶表达载体ｐＴ７ｚｚ,得到表达质粒ｐＦｕ,经转化至表达宿主Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）,在ＩＰＴＧ诱导下获得融合蛋白目的产物的直接表达。经ＳＤＳ－ＰＡＧＥ电泳鉴     

抗真菌蛋白Rs—AFPs基因在大肠杆菌中的表达

将抗真菌蛋白Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２全长ｃＤＮＡ插入表达质粒ｐＥＴ－２２ｂ／ＮｃｏＩ＋ＳａｃＩ位点,构建成融合蛋白表达载体ｐＲＡＦ１和ｐＲＡＦ２．将不含信号肽编码序列的Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２ｃＤＮＡ分别插入ｐＥＴ－２２ｂ／Ｎｃｏｌ＋Ｓａｃｌ和ｐＥＴ－２２ｂ／Ｎｄｅｌ＋ＳａｃＩ位点,构建成不含信号肽序列的融合蛋白表达载体ｐＲＡＦ３、ｐＲＡＦ４和非融合蛋白表达载体ｐＲＡＦ５和ｐＲＡＦ６．将构建的上述各种表达载体转化Ｅ．ｃｏｌｉＢＬ２１,挑菌落培养,ＩＰＴＧ诱导,使Ｒｓ－ＡＦＰｓ基因得到表达,并用体外抑菌试验检测表达产物的活性,结果表明,各种表达载体的表达产物均具有不同程度的抑菌活性,其中,ｐＲＡＦ３和ｐＲＡＦ４表达产物的抑菌活性较明显．     

乙型肝炎、肝硬变和肝癌中HBxAg的表达及其在肝癌发生中的作用

对３７９例良、恶性肝组织进行的免疫组织化学研究显示,３３％的慢性迁延性肝炎（６／１８）、７６％的慢性活动性肝炎（２６／３４）、９２％的肝硬变（５７／６２）和９７％的肝细胞性肝癌（ＨＣＣ）（５８／６０）中有ＨＢｘＡｇ表达,阳性率高于ＨＢｓＡｇ或ＨＢｃＡｇ。癌周肝中的ＨＢｘＡｇ阳性率显著高于非癌周肝。与其它２种ＨＢＶ抗原不同,ＨＢｘＡｇ表达在细胞类型上有较明显的选择性,在肝小多角细胞（ＳＰＬＣ）、小细胞性不典型增生（ＳＣＤ）及ＨＣＣ中较强。与ＩＧＦⅡ、ｃ－ｅｒｂＢ－２、ｃ－ｍｙｃ和ＥＧＦ－Ｒ表达进行的对照研究表明ＨＢｘＡｇ与ＩＧＦⅡ和ｃ－ｅｒｂＢ－２这２种ＨＣＣ发生相关基因的表达关系密切。ＰＣＮＡ染色结果显示ＨＢｘＡｇ阳性组织的细胞增殖活性显著高于ＨＢｘＡｇ阴性组织。我们的结果还表明ＨＢｘＡｇ表达与肝细胞不典型增生的发生和进展有关、提出ＨＢＶＸ基因可能通过其表达产物（ＨＢｘＡｇ）首先激活ＩＧＦⅡ、ｃ－ｅｒｂＢ－２基因,继而引起显著的ＳＰＬＣ增生和ＳＣＤ而参与ＨＣＣ发生的．     

乙型肝炎病毒前S2抗原决定簇（HBVPreS2epitope）与乙型肝炎病毒核心抗原（HBcAg）的基因融合与表达

乙肝前Ｓ２（ＨＢＶＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇。我们将化学合成的ＰｒｅＳ２ｅｐｉｔｏｐｅ（１２０－１４５）基因与ＨＢｃＡｇ基因不同位点进行融合,融合基因在大肠杆菌中获得表达,并对融合蛋白进行了纯化。经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明,融合蛋白具有ＰｒｅＳ２和ＨＢｃＡｇ两者的抗原性。此外,研究还表明,强启动子能使表达水平有一定提高。     

绿色荧光蛋白基因在青蒿转基因芽中的表达

将改良的绿色荧光蛋白（ＧＦＰ）基因,插入到植物表达载体中,构建双ＣａＭＶ３５Ｓ启动子驱动下的植物表达载体ｐＢＩＧＦＰ,在Ｋａｍ浓度为２０ｍｇ／Ｌ的筛选培养基上,用含有ｐＢＩＦＰ质粒的根癌农杆菌ＬＢＡ４４０４感染青蒿叶片,获得５个抗Ｋａｎ阳性丛生芽系。Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ分析表明,外源ＧＦＰ基因已整合到青蒿转基因芽Ｇ－１系的基因组中。在ＯＬＹＭＰＵＳ－ＢＨ２型荧光显微镜下,观察到转基因     

重组人融合基因Nm23—H1／HbFGF的构建及其在大肠杆菌中表达？…

根据Ｎｍ２３－Ｈ１与ＨｂＦＧＦｃＤＮＡ序列,人工合成一段中间核酸序列,将它分别与Ｎｍ２３－Ｈ１ｃＤＮＡ的上游引物及ＨｂＦＧＦｃＤＮＡ的下游引物组成两对引物,通过ＰＣＲ（聚合酶链式反应）构建出融合基因Ｎｍ２３－Ｈ１／ＨｂＦＧＦ,将其定向克隆于质粒载体ｐＢＶ２２０上,经诱导,ＳＤＳ－ＰＡＧＥ分析,表达产物分子量为３４ｋＤ,表达量占菌体总蛋白的１４％,表达产物以包涵体形式存在,ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ     

乙型肝炎病毒e抗原基因与绿色荧光蛋白基因的融合及其表达

质粒pS65T含有T7启动子驱动的绿色荧光蛋白突变型gfpS65T基因,经修饰后,在其中插入乙型肝炎病毒e抗原（HBeAg）基因,使两基因同框成为融合基因。在大肠杆菌BL21中由于T7启动子的控制,高效表达了具有双功能（抗原性和发光性）的融合蛋白（GFP－HBeAg）。融合蛋白MW为52kDa,N端有6个组氨酸残基,因而用Ni金属螯合层析柱对融合蛋白进行了分离纯化,利用诊断HBV的ELISA试剂盒检测了融合蛋白的抗原性,在荧光显微镜下观察到了融合蛋白的绿色荧光。并探讨了用其组装成新型免疫诊断试剂的可能性。     

Preparation and Properties of Clostridium thermocellum Lichenase Deletion Variants and Their Use for Construction of Bifunctional Hybrid Proteins

Major properties (pH and temperature optimum, stability) of lichenase (b-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.     

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

绿色荧光蛋白融合人核糖核酸酶抑制因子表达载体pEGFP-C1-hri的构建及鉴定

目的构建表达绿色荧光蛋白融合人核糖核酸酶抑制因子的表达载体pEGFP—C1—hri,为探讨人核糖核酸酶抑制因子抗肿瘤作用的分子机制打下基础。方法用亚克隆法,将目的片段从表达载体pGEX-6p-1-hri克隆到pEGFP-C1上,用双酶切筛选得到阳性重组质粒pEGFP—C1—hri,用脂质体法将其瞬时转染到小鼠黑色素瘤细胞B16中,在荧光显微镜下检测其表达。结果pEGFP—C1—hri中插入了hri序列,绿色荧光高效表达于B16细胞浆中。结论pEGFP—C1—hri表达载体已成功构建。     

玉米大斑病菌STK1与EGFP融合基因载体的构建及其在毕赤酵母中的表达

STK1基因是玉米大斑病菌调控分生孢子发育、渗透胁迫调节和致病性的重要MAPK基因。本文首先构建了含有增强型绿色荧光蛋白基因(EGFP)的毕赤酵母GSS115(Pichia pastoris GS115)表达载体p PIC3.5K-EGFP,再以玉米大班病菌模式菌株01-23的菌丝c DNA为模板,PCR扩增STK1基因,克隆到p PIC3.5K-EGFP,构建了STK1-EGFP融合基因的GS115表达载体p PIC3.5K-STK1-EGFP。利用电击转化法将该融合基因表达载体转化到GS115感受态细胞内,利用MD培养基筛选、PCR鉴定,获得了STK1-EGFP融合基因的毕赤酵母转化子。通过RT-PCR和荧光观察,发现STK1基因和EGFP基因均可以高效稳定地表达。另外,在试验中我们还发现,在STK1基因起始密码子前加入Kozak序列可以使STK1-EGFP融合基因的表达强度增强4.8倍。以上研究结果为STK1基因表达蛋白的亚细胞功能定位和抗体制备奠定了基础。     

Expression and purification of homogenous proteins in Saccharomyces cerevisiae based on ubiquitin-FLAG fusion

The construction of an expression vector for increased expression of cytoplasmic proteins in Saccharomyces cerevisiae is described. To enhance the yield of expressed proteins, fusion of ubiquitin to an octapeptide (a FLAG tag) upstream of the respective model genes was applied. During protein maturation ubiquitin is efficiently removed by yeast autologous hydrolases, generating the FLAG octapeptide at the N-terminus. Fusion proteins were recognized by the specific monoclonal antibody M1 directed against the FLAG tag. The FLAG-tagged proteins were purified to homogeneity by immunoaffinity chromatography using an anti-FLAG M1 agarose. Different model proteins, green fluorescent protein, green fluorescent protein-human lysozyme, green fluorescent protein elongation-initiahon factor 5a, green fluorescent protein-rapamycin-selective 25-kDa immunophilin, and green fluorescent protein-heat shock protein 90 beta have been selected to demonstrate the efficiency of the new vector construct.     

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii†

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.