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Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(4):646-649
In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white. 相似文献
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目的:通过多点突变构建增强型青色荧光蛋白(ECFP)慢病毒表达载体。方法与结果:根据增强型绿色荧光蛋白(EGFP)和ECFP基因序列的差异设计3对引物,以pLentiLox3.7-EGFP为模板进行分段PCR扩增,再以分段PCR扩增产物为模板扩增出突变的ECFP基因片段,将其与载体连接,得到ECFP慢病毒表达载体pLentiLox3.7-ECFP,测序结果证实经过多点突变扩增的ECFP片段基因序列完全正确;磷酸钙介导pLentiLox3.7-ECFP在293T细胞中表达,48h后在荧光显微镜下观察到青色荧光蛋白。结论:通过多点突变的方法得到了ECFP慢病毒表达载体。 相似文献
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Streptococcus suis serotype 2 (S. suis 2) is an important pathogen, responsible for diverse diseases in swine and humans. To obtain a S. suis 2 strain that can be tracked in vitro and in vivo, we constructed the Egfp-HA9801 recombinant S. suis 2 strain with egfp and spc(r) genes inserted via homologous recombination. To assess the effects of the egfp and spc(r) genes in HA9801, the biochemical characteristics, growth features and virulence in Balb/C mice were compared between the recombinant and the parent HA9801 strain. We detected the EGFP expression from Egfp-HA9801 by epifluorescence microscopy. The results showed that the biochemical characterization and growth features of the Egfp-HA9801 recombinant were highly similar to that of the parent HA9801. We did not find significant differences in lethality (50% lethal dose), morbidity and mortality between the two strains. Furthermore, the bacterial counts in each various tissues of Egfp-HA9801-infected mice displayed similar dynamic compared with the HA9801-infected mice. Our results also showed that the Egfp-HA9801 cells grown at 37°C for 36 h displayed greater green fluorescence signals than the cells grown at 28°C for 36 h and 37°C for 24 h. The fluorescence in the tissue cryosections of Egfp-HA9801-injected mice was also stronger than that of the HA9801 group. Together, these results indicate that the egfp and spc(r) insertions into the Egfp-HA9801 recombinant did not significantly change the virulence when compared with HA980, and this EGFP labeled strain can be used for future S. suis 2 pathogenesis research. 相似文献
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表达绿色荧光蛋白突变体的重组伪狂犬病毒的构建及其增殖特性 总被引:2,自引:1,他引:2
将绿色荧光蛋白突变体M 1(EGFPS14 7/P)基因融合到伪狂犬病毒 (PRV)非必需糖蛋白 gG的第 8个氨基酸下游 ,通过同源重组、空斑纯化和PCR筛选获得能表达M 1并导致gG基因部分缺失的重组病毒 gG-/M1 。重组病毒经Southern杂交、Western印迹和荧光观察证实构建正确。纯化的重组病毒以低感染指数接种PK 15细胞 ,在感染早期 (6h)就能观察到荧光 ,随着病毒的增殖 ,荧光逐渐增强 (2 4~ 36h) ,直至完全病变 ,荧光淬灭。进一步对重组病毒gG-/M1 与亲本株gG-/LacZ 、野毒株的增殖特性进行比较 ,发现 3种毒株在增殖滴度上无显著差异。上述结果表明构建的PRVgG-/M1 突变株能作为活细胞示踪实时监测病毒感染的动态分析。 相似文献
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Zifu Zhang Peng Sun Fuxian Yu Li Yan Fang Yuan Wenxin Zhang Tao Wang Zhiyi Wan Qiang Shao Zandong Li 《PloS one》2012,7(12)
Several strategies have been used to generate transgenic birds. The most successful method so far has been the injection of lentiviral vectors into the subgerminal cavity of a newly laid egg. We report here a new, easy and effective way to produce transgenic quails through direct injection of a lentiviral vector, containing an enhanced-green fluorescent protein (eGFP) transgene, into the blood vessels of quail embryos at Hamburger-Hamilton stage 13–15 (HH13–15). A total of 80 embryos were injected and 48 G0 chimeras (60%) were hatched. Most injected embryo organs and tissues of hatched quails were positive for eGFP. In five out of 21 mature G0 male quails, the semen was eGFP-positive, as detected by polymerase chain reaction (PCR), indicating transgenic germ line chimeras. Testcross and genetic analyses revealed that the G0 quail produced transgenic G1 offspring; of 46 G1 hatchlings, 6 were transgenic (6/46, 13.0%). We also compared this new method with the conventional transgenesis using stage X subgerminal cavity injection. Total 240 quail embryos were injected by subgerminal cavity injection, of which 34 (14.1%) were hatched, significantly lower than the new method. From these hatched quails semen samples were collected from 19 sexually matured males and tested for the transgene by PCR. The transgene was present in three G0 male quails and only 4/236 G1 offspring (1.7%) were transgenic. In conclusion, we developed a novel bird transgenic method by injection of lentiviral vector into embryonic blood vessel at HH 13–15 stage, which result in significant higher transgenic efficiency than the conventional subgerminal cavity injection. 相似文献
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In Vivo Tracking of Campylobacter jejuni by Using a Novel Recombinant Expressing Green Fluorescent Protein 下载免费PDF全文
Philip F. Mixter John D. Klena Gary A. Flom Amy M. Siegesmund Michael E. Konkel 《Applied microbiology》2003,69(5):2864-2874
Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b+ Gr-1+ lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+ Gr-1− lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b− CD45R+ B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate that C. jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C. jejuni genotypic variants can be isolated from both in vitro and in vivo systems. 相似文献
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目的:构建携带人脆性X相关基因1(Fragile X related gene 1,FXR1)与增强型绿色荧光蛋白(Enhanced green fluorescent pro-tein,EGFP)的融合表达载体并进行表达,为研究FXR1基因在脆性X综合征中的作用机制奠定基础.方法:以重组质粒pYESTrp3/FXR1为模板,PCR扩增FXR1基因,将扩增片段双酶切后连接到质粒pEGFP-N1中,形成重组表达载体pEGFP-N1/FXR1,并用脂质体法转染人胚肾细胞,荧光显微镜下观察GFP在细胞内的表达及Western Blotting检测FXR1的表达情况.结果:成功构建融合表达载体pEGFP-N1/FXR1,在人胚肾细胞实现表达,检测到融合蛋白EGFP/FXR1.结论:本实验成功构建EFGP和FXR1重组共表达载体并可在真核细胞中表达,为进一步研究FXR1在脆性X综合征中的作用机理奠定基础. 相似文献
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A new bacterial cloning vector, pGreenLD, derived from the triple substitution mutated Aequorea v/ctor/a green fluorescent protein(GFP-S65A, V68L, S72A), when expressed in E. coli produced colonies which showed yellow-green colour under daylight and strong green fluorescence under long-wave ultraviolet light. It can be a useful vector for selecting foreign DNA fragment which was inserted into multiple cloning site based on the loss of the yellow-green color/green fluorescence of E. coli cells attributable to the insertional inactivation of GFP production. 相似文献
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目的构建和鉴定HAX1和EGFP双基因共表达重组腺病毒载体。方法采用DNA重组技术,将目的基因HAX1克隆至含有报告基因EGFP的穿梭质粒pAdTrack—CMV中,并转化于大肠埃希菌DH5a;筛选出重组质粒pAdTrack—CMV—HAX1,并在BJ5183细菌中与pAdEasy-1质粒进行同源重组,产生重组腺病毒载体;用lipofectamine将其转染HEK293细胞,包装携带全长HAX1的重组复制缺陷型腺病毒pad—HAX1-EGFP,酶切和序列测定鉴定;用制备好的Ad—HAX1-EGFP感染HEK293细胞,流式细胞术检测其感染效率,RT—PCR、Western印迹鉴定外源基因HAX1的表达。BrdU检测感染了Ad—HAX1-EGFP的HEK293细胞增殖情况。结果pAdTrack—CMV—HAX1重组质粒构建成功。pAdTrack—CMV—HAX1质粒与pAdEasy-1质粒同源重组后与预期结果相符。构建好的Ad—HAX1-EGFP能有效感染HEK293细胞;外源基因能在239细胞中有效表达。HAX1高表达的HEK293细胞其增殖率得以提高。结论成功构建了表达HAX1和EGFP共表达的重组腺病毒载体,HAX1能够促进结肠癌细胞HEK293细胞的增殖。 相似文献
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绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。 相似文献
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为对重组蛋白的表达进行直观检测并简化蛋白纯化的步骤,构建了能在大肠杆菌中表达融合蛋白的通用表达载体pHis-EGFP。该载体含有源自表达载体pET-32a的T7启动子、终止子和源自质粒pUC18的ColE1复制子与绿色荧光蛋白报告基因。应用该载体成功地表达并纯化了酵母GGDP(geranylgeranyldiphosphate,GGDP)合酶融合蛋白,结果表明所构建的载体是一个实用的表达载体,并建立了离子交换层析和亲和层析两步纯化融合蛋白的方法。 相似文献