对虾白斑综合征病毒ORF220基因在昆虫细胞内的表达及其分布

对虾白斑综合征病毒厦门分离株ORF220编码真核生物GP130受体同源蛋白.将ORF220和绿色荧光蛋白编码基因融合在一起克隆到昆虫杆状病毒表达载体pFastBacI,然后与AcBacmid共同转染DH10B细胞.用PCR鉴定含有ORF220和EGFP基因的重组质粒,提取纯化重组质粒并转染昆虫细胞进行表达.结果发现,DNA转染后3-5d可以在荧光显微镜下观察到绿色荧光,表明融合蛋白在昆虫系统内成功表达.用病毒上清液感染昆虫细胞进行时相观察,结果表明,ORF220蛋白在昆虫细胞的细胞质和细胞核内呈随机分布,没有特异的细胞定位.     

pEGFP—HPV16E7重组融合蛋白质粒的构建及其表达

应用基因重组技术,构建增强绿色荧光蛋白(EGFP)与人乳头瘤病毒16型E7(HPV16E7)的重组融合表达质粒,经限制性内切酶酶切鉴定和PCR分析后,用基因转染技术将其导入小鼠肝癌细胞,荧光显微镜下观察融合蛋白的表达。酶切鉴定和PCR分析证实重组质粒中插入目的基因片段的大小、方向和插入位点均正确,在转染的小鼠肝癌细胞中观察到绿色荧光蛋白的表达。构建的pEGFP-HPV16E7融合表达质粒能直观地反映转染细胞中EGFP-HPV16E7融合蛋白的表达。由于转化率与表达率融为一体,故有利于对转染细胞的筛选,缩短转染细胞在体外的筛选的时间适用于对：HPV16E7分子生物学特性．致瘤机理及APC提呈等的研究。为建立表达HPV16E7的实体瘤动物模型奠定了基础。     

pEGFP-HPV16E7重组融合蛋白质粒的构建及其表达

应用基因重组技术,构建增强绿色荧光蛋白(EGFP)与人乳头瘤病毒16型E7(HPV16E7)的重组融合表达质粒,经限制性内切酶酶切鉴定和PCR分析后,用基因转染技术将其导入小鼠肝癌细胞,荧光显微镜下观察融合蛋白的表达.酶切鉴定和PCR分析证实重组质粒中插入目的基因片段的大小、方向和插入位点均正确,在转染的小鼠肝癌细胞中观察到绿色荧光蛋白的表达.构建的pEGFP-HPV16E7融合表达质粒能直观地反映转染细胞中EGFP-HPV16E7融合蛋白的表达.由于转化率与表达率融为一体,故有利于对转染细胞的筛选,缩短转染细胞在体外的筛选的时间适用于对HPV16E7分子生物学特性、致瘤机理及APC提呈等的研究.为建立表达HPV16E7的实体瘤动物模型奠定了基础.     

大鼠大麻素Ⅰ型受体绿色荧光融合蛋白真核表达载体的构建与鉴定

目的:构建大鼠大麻素型Ⅰ受体绿色荧光融合蛋白真核表达载体并观察其在细胞中的表达。方法:大鼠CB1基因序列设计引物,以大鼠脑组织为模板扩增CB1基因编码区片段,克隆至增强型绿色荧光蛋白表达载体pEGFP-N3中,构建重组融合蛋白表达载体pCB1-EGFP。将pCB1-EGFP质粒转染HeLa细胞,通过观察EGFP报告基因的表达以及免疫荧光,Western Blot方法鉴定CB1可在真核细胞中过表达情况。结果:构建重组融合蛋白表达载体pCB1-EGFP,单双酶切和测序验证正确。将pCB1-EGFP质粒转染HeLa细胞,荧光显微镜下观察到融合表达的绿色荧光蛋白,且呈胞膜表达。免疫荧光试验也证明重组载体转染后,CB1基因和GFP共同定位于胞膜部分。Western Blot实验证明表达CB1蛋白。结论:成功构建了高表达的CB1-EGFP融合蛋白真核表达载体。     

携带EGFP基因的戊型肝炎病毒重组质粒的构建及其感染性的验证

为了构建携带增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的戊型肝炎病毒(Hepatitis E virus,HEV)重组质粒,转染人肺癌细胞A549细胞验证其感染性,PCR法分两段扩增HEV全基因组序列和EGFP基因序列,将EGFP报告基因插入到HEV ORF2基因下游,并克隆到体外转录表达载体pGEM-7Zf(+)上。然后利用脂质体转染法将重组质粒转入A549细胞,24h后在荧光显微镜下观察EGFP的表达;转染72h后,利用免疫荧光法检测HEV ORF2蛋白的表达。转染7d后将发生病变的A549细胞收集作为接种物,接种A549细胞验证携带EGFP的HEV-EGFP重组病毒的感染性。结果显示经酶切和测序鉴定携带EGFP的HEV重组表达质粒pGEM-HEV-EGFP构建成功;EGFP基因与HEV在A549细胞中可融合表达;携带EGFP的HEV重组表达质粒转染A549细胞7d后出现病变,并且连续传代3代仍具有感染性。本研究成功构建了携带EGFP的HEV全基因组重组质粒pGEM-HEV-EGFP,并成功感染A549细胞,为进一步研究HEV的复制机制及致病机理奠定基础。     

SDF-1α基因与绿色荧光蛋白融合载体的构建及亚细胞定位

目的构建SDF-1α基因与绿色荧光蛋白的融合蛋白表达载体,进而观察SDF-1α基因编码蛋白在细胞内的定位情况。方法用EcoRI内切酶从pMD-T18一SDF-1α重组载体中酶切分离SDF-1α基因的完整ORF,构建pEGFP-C1-SDF-1α的融合表达载体,脂质体转染COS-7细胞,并在荧光显微镜下观察表达的融合蛋白。结果SDF-1α基因在COS-7细胞中高效表达,激光共聚焦的结果显示,SDF-1α基因定位在细胞质内。结论成功构建了pEGFP-C1-SDF-1α的融合表达载体,SDF-1α基因主要在细胞质中表达。     

日本鳗鲡绿色荧光蛋白基因的克隆与表达

[目的]克隆日本鳗鲡绿色荧光蛋白UnaG基因,表达并纯化UnaG蛋白。[方法]从日本鳗鲡中克隆了绿色荧光UnaG基因,并克隆至p ET28Aa、pc DNA-Flag质粒中。将重组质粒pc DNA-Flag用脂质体转染到293T细胞中,用荧光显微镜观察绿色荧光,同时用原核表达系统诱导His-UnaG融合蛋白的表达,用亲和层析和凝胶过滤层析纯化HisUnaGg融合蛋白。[结果]转染人293T细胞后不到24 h,荧光显微镜观察到日本鳗鲡UnaG能被激发出较强的绿色荧光;原核表达并纯化的His-UnaG融合蛋白纯度很高,Western Blot检测为单一条带,蛋白量达3μg/μl。[结论]成功克隆并纯化了UnaG,为进一步研究UnaG的应用奠定基础。     

MIC3-EGFP融合蛋白真核表达质粒的构建及其在COS-7细胞中的表达

目的:构建以绿色荧光蛋白(greenfluoreseeneeprotein,GFP)为报告基因的重组表达质粒pEGFP-C2-MIC3并检测MIC3-EGFP融合蛋白其在COS-7细胞中的表达及定位.方法:通过基因重组的方法构建pEGFP-C2-MIC3重组真核表达质粒,并通过酶切和基因测序鉴定.脂质体法转染体外培养的COS-7细胞,转染后24h在活细胞状态下用倒置荧光显微镜直接观察MIC3-EGFP融合蛋白在COS-7细胞中的分布.结果:PCR检测,酶切鉴定及测序证实目的基因MIC3正确连接到pEGFP-C2的多克隆位点.pEGFP-C2-MIC3重组体转染COS-7后,在细胞质表达.结论:成功地构建了pEGFP-C2-MIC3融合蛋白真核表达质粒,在COS-7细胞中获得表达.     

细菌内同源重组法制备FMDV聚蛋白编码基因重组腺病毒

采用PCR方法从重组质粒pMD18_T/PP中扩增出FMDV的聚蛋白(PP)编码基因,再亚克隆至腺病毒穿梭质粒中,形成重组穿梭质粒rpAd_CMV/PP;将获得的重组穿梭质粒与腺病毒骨架载体通过在大肠杆菌内质粒间同源重组获得重组腺病毒质粒rpAd/PP。将腺病毒载体线性化后用脂质体介导转染293细胞从而获得含有口蹄疫病毒PP编码基因的重组腺病毒。通过倒置显微镜观测,可见明显的细胞病变,利用荧光显微镜可观测到报告基因绿色荧光蛋白的表达,并在电镜下观察到FMDV的空衣壳。结果证明已成功获得了含有口蹄疫病毒PP编码基因的重组腺病毒rAd/PP,并成功表达组装FMDV空衣壳,为FMDV腺病毒活载体疫苗的研究奠定了基础。     

HSV-2LAT ORF1在Vero细胞中的表达特性及其对细胞活性的影响

目的:研究单纯疱疹病毒Ⅱ型(HSV-2)潜伏相关转录体(LAT)开放读码框1(ORF1)的表达特点及其对Vero细胞活性的影响.方法:双酶切和测序验证本实验室构建的HSV-2 LAT ORF1真核表达载体pEGFP-ORF1,并以转染试剂盒Xfect介导其转染至Vero细胞,通过RT-PCR和绿色荧光蛋白检验其在细胞中的表达,用MTT法进行细胞活性分析.结果:重组质粒表达的融合蛋白主要集中细胞核,而空质粒表达的绿色荧光蛋白在细胞核和细胞质中分布均匀;重组质粒对Vero细胞没有损伤作用.结论:HSV-2 LAT ORF1影响了绿色荧光蛋白的分布,可降低空质粒对细胞的损伤作用;其作用位点可能主要定位在细胞核中,为阐明HSV-2 LAT ORF1在潜伏复发中的功能奠定了实验基础.     

Expression of SLAM (CDw150) on Sf9 cell surface using recombinant baculovirus mediates measles virus infection in the nonpermissive cells

Signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been reported as the receptor of measles virus (MV) interacting with MV hemagglutinin (MVH). In this study, we developed a baculovirus-derived vector, the Bacmid-egfp, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the control of the promoter of very late polyhedrin gene from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and employed the recombinant baculovirus to express SLAM in Sf9 (Spodoptera frugiperda) cells and investigate SLAM function. The result showed that the integration of the EGFP expression cassette in the Bac-to-Bac system facilitated research with the system without introducing compromises due to its use. SLAM protein fused to His-tag was expressed in Sf9 cells through the modified Bac-to-Bac system. The expressed SLAM was identified as approximately 46 kDa, and it presented on the cell surface, as revealed by fluorescent immunochemical staining and confocal microscopic analysis. The pull-down assay proved that SLAM protein expressed in this system could interact with MVH protein. After incubating with MV vaccine strain S191, cell fusion was only observed in the Sf9 cells expressing both EGFP and SLAM from recombinant baculovirus rather than those expressing EGFP only from the modified viral vector. Furthermore, MV replicated and induced apoptosis in the Sf9 cells with SLAM expression.     

A surface-modified baculovirus vector with improved gene delivery to B-lymphocytic cells

A short peptide motif from gp350/220 of Epstein-Barr virus, EDPGFFNVEI, which was known to bind to CD21, a surface protein on B-lymphocyte, was inserted into the baculovirus surface protein gp64. The recombinant virus carrying the hybrid gp64/gp350 gene, vAc-gp350EGFP, was obtained, and the expression of gp64/gp350 protein was confirmed with immunoblot using anti-gp350 antibody. When compared with a control virus with wild type gp64, vAc-gp350EGFP showed increased transduction efficiency in B cell lines Raji, HR1, B95-8, BJAB, and DG75, regardless of their being EBV-positive or EBV-negative. No such increase was seen in non-B cell lines HEK293 and HeLa. When Raji cells were transduced with increased amount of vAc-gp350EGFP, transduction became saturated when the multiplicity of infection was higher than 20pfu/cell. The transduction of Raji cells by vAc-gp350EGFP was dose-dependently inhibited by pre-treatment of cells with anti-CD21 antibody. These results showed that vAc-gp350EGFP entered B cells by interacting with CD21.     

ORF390 of white spot syndrome virus genome is identified as a novel anti-apoptosis gene

Apoptosis serves as an important defense strategy employed by host cells against viral invasion. Many viruses contain the anti-apoptotic genes to block the defense-by-death response of host cells. In this study, we tried to identify the putative anti-apoptotic genes in white spot syndrome virus (WSSV) genome. We confirmed that actinomycin D could induce apoptosis of shrimp primary cells. However, the apoptosis triggered by actinomycin D was inhibited by WSSV infection. As mutants of Autographa californica nucleopolyhedrovirus (AcMNPV), AcMNPVDelta35k/pol+ lacks a functional P35 gene undergoing apoptosis and its infection could induce Sf9 cell apoptosis. To identify the putative apoptotic suppressor gene of WSSV, overlapping cosmid clones representing the entire WSSV genome were individually cotransfected along with genome DNA of AcMNPVDeltaP35k/pol+. Using this marker rescue assay, a WSSV DNA fragment that was able to rescue AcMNPVDeltaP35k/pol+ infection in Sf9 cells was isolated. By further sequence analysis and rescue assay, the ORF390 was identified as a novel anti-apoptotic gene. The ORF displays two putative caspase9 cleavage sites LLVETDGPS, VKLEHDGSK, and a caspase3 cleavage site EEDEVDGVP. The ORF was cloned into the pIE1 vector and then the recombinant vector was transfected into Sf9 cells. The Sf9 cells did not show obvious characteristics of apoptosis when infected with AcMNPVDeltaP35k/pol+. And the transient expression of ORF390 allowed AcMNPVDeltaP35k/pol+ replication in Sf9 cells and resulted in the formation of polyhedra successfully. The results indicate that function of ORF390 in WSSV is a kind of apoptotic suppressor like P35 in AcMNPV.     

Simple Purification of a Foreign Protein Using Polyhedrin Fusion in a Baculovirus Expression System

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures, and that these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with the Polh gene at the N-terminus, including a linker and enterokinase (EK) site between Polh and EGFP, was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, EGFP presented mainly in the supernatant, and this supernatant fraction also showed a pure EGFP band. These results suggest that a combined procedure of Polh fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

增强型绿色荧光蛋白的真核表达和纯化

根据编码增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的开放读码框(open reading frame,ORF)设计引物,PCR方法扩增出5'端带His标签的EGF PORF,利用杆状病毒表达系统构建表达EGFP基因的重组杆状病毒DNA分子,转染sf9细胞.取细胞...     

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway.

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

Sema4C及其相互作用蛋白GIPC的亚细胞定位及荧光共定位研究

目的：观察脑信号蛋白Sema4C及其相互作用蛋白GIPC的亚细胞定位及两者的荧光共定位情况,为明确Sema4C和GIPC在亚细胞水平的相互作用提供佐证。方法：将Sema4C的基因编码区全长、胞外段和胞内段分别构建到pEGFPNl和pEGFPCI表达载体中,将GIPC编码区基因构建到pDsRed-C1表达载体中,分别转染HEK293细胞,观察亚细胞定位;将pEGFPNl-Sema4C和pDsRed-GIPC分别共转染HEl（293和COS7细胞,观察两者的荧光共定位情况。结果：酶切鉴定及测序结果表明重组载体构建正确,Sema4C蛋白全长和胞外段呈跨膜分布,而胞内段在全细胞中呈弥散样分布;GIPC在胞浆内呈斑块状聚集分布;pEGFPNl-Sema4C和pDsRed-GIPC存在荧光共定位区域。结论：Sema4C主要在胞膜和胞浆内表达,GIPC主要在胞浆内呈斑块样聚集分布;Sema4C和GIPC之间存在荧光共定位。     

Plasma phospholipid transfer protein fused with green fluorescent protein is secreted by HepG2 cells and displays phosphatidylcholine transfer activity.

Phospholipid transfer protein (PLTP) is a serum glycoprotein with a central role in high-density lipoprotein metabolism. We created a fusion protein in which enhanced green fluorescent protein (EGFP) was fused to the carboxyl-terminus of PLTP. Stably transfected HepG2 cells, which overexpress this fusion protein, were generated. PLTP-EGFP was translocated into the ER and fluoresced within the biosynthetic pathway, showing a marked concentration in the Golgi complex. The transfected cells secreted into the growth medium phospholipid transfer activity 7-fold higher than that of the mock-transfected controls. The medium of the PLTP-EGFP - expressing cells displayed EGFP fluorescence, demonstrating that both the PLTP and the EGFP moieties had attained a biologically active conformation. However, the specific activity of PLTP-EGFP in the medium was markedly reduced as compared with that of endogenous PLTP. This suggests that the EGFP attached to the carboxyl-terminal tail of PLTP interferes with the interaction of PLTP with its substrates or with the lipid transfer process itself. Fluorescently tagged PLTP is a useful tool for elucidating the intracellular functions of PLTP and the interaction of exogenously added PLTP with cells, and will provide a means of monitoring the distribution of exogenously added PLTP between serum lipoprotein subspecies.     

HSV-2 LAT ORF2在Vero细胞中的表达及对细胞的作用

旨在研究人单纯疱疹病毒2型(HSV-2)潜伏相关转录体(LAT)开放读码框2(ORF2)对体外培养的非洲绿猴肾细胞(Vero)形态和活性的作用。将构建好的带绿色荧光蛋白(EGFP)标签的HSV-2LAT ORF2真核表达载体pEGFP-ORF2,转染vero细胞,荧光显微镜观测细胞形态的改变和MTT法进行活性分析。结果:显示,HSV-2LATORF2诱导细胞形态发生明显变化,绿色荧光蛋白在细胞的定位也发生了改变,细胞活性降低。由此证实,HSV-2LAT ORF2对细胞有损伤作用,为阐明HSV-2LATORF2的功能提供了资料。