人P2X7真核表达载体的构建及稳定转染细胞株的建立

目的：构建人P2X7基因的真核表达载体,并通过转染获得稳定表达P2X7分子的HEK293细胞株。方法：以人脑组织P2X7cDNA为模板扩增出P2X7基因,插入到真核表达载体pEGFP-N1中,构建重组质粒pEGFP-N1/P2X7。用X-fect试剂盒将重组质粒转染HEK293细胞,通过G418辅助荧光筛选建立稳定表达P2X7-EGFP细胞株。经流式细胞仪、Western blot和激光共聚焦显微镜检测,了解人P2X7在HEK293细胞中的表达水平及细胞内定位。结果：重组质粒pEGFP-N1/P2X7构建正确,建立了稳定表达人P2X7的HEK293细胞系。Western blot和流式细胞仪检测证实,P2X7在HEK293细胞系中成功表达,激光共聚焦显微镜检测显示P2X7-EGFP定位在细胞膜上。结论：重组载体pEGFP-N1/P2X7构建成功并建立了稳定表达人P2X7的HEK293细胞系,为进一步研究P2X7离子通道结构和功能奠定基础。     

BTI基因转染诱导EC9706细胞凋亡及G_1阻滞

为了研究荞麦胰蛋白酶抑制剂(buckwheat trypsin inhibitor,BTI)对肿瘤细胞凋亡与细胞周期的影响,构建增强型绿色荧光蛋白(EGFP)与BTI融合蛋白真核表达质粒.将BTI基因成功克隆至pEGFP-N1中转染食管癌EC9706细胞后,激光共聚焦显微镜镜检显示,BTI-EGFP获得良好表达.表达的融合蛋白大部分分布于细胞核,在细胞质中有少量分布.Western印迹检测可见约27kD和36 kD的特异性条带.流式细胞术分析结果显示,BTI能够诱导EC9706细胞发生凋亡,并使细胞停滞于G0/G1期.     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

一种基于EGFP的、安全的抗HIV药物评价系统

目的：建立基于EGFP的、安全的抗人免疫缺陷病毒（HIV）药物评价系统。方法：用增强型绿色荧光蛋白（EGFP）基因替代HIV感染性克隆质粒pUC18-HIV-NL4-3中的部分包膜基因（env）,构建重组假病毒质粒pUC18-NL4-3-EGFP,将其与水疱性口炎病毒糖蛋白（VSV-G）真核表达载体共转染人胚肾293FT细胞,观察绿色荧光蛋白的表达,同时用该细胞培养上清进一步感染其他293FT细胞培养物。为了检验该假病毒系统能否用于抗病毒药物的评价,在假病毒复制和感染过程中加入不同浓度的抗HIV药物AZT（Zidovudine）,采用荧光显微镜检测和流式细胞仪定量检测,分析AZT对假病毒的抑制作用。结果：假病毒质粒pUC18-NL4-3-EGFP能够在转染细胞和再感染细胞中有效地表达绿色荧光蛋白基因,不同浓度的AZT能以剂量依赖方式抑制假病毒的感染和报告基因的表达。结论：建立了一种基于EGFP表达和检测的、安全的HIV假病毒复制和感染系统,该系统可以用于抗HIV药物的筛选和评价。     

Electrophysiological and Fluorescence Microscopy Studies with HERG Channel/EGFP Fusion Proteins

HERG (human ether-a-go-go-related gene) encodes the Kv11.1 protein α-subunit that underlies the rapidly activating delayed rectifier K⁺ current (I _Kr) in the heart. Alterations in the functional properties or membrane incorporation of HERG channels, either by genetic mutations or by administration of drugs, play major roles in the development of life-threatening torsades de pointes cardiac arrhythmias. Visualization of ion channel localization is facilitated by enhanced green fluorescent protein (EGFP) tagging, but this process can alter their properties. The aim of the present study was to characterize the electrophysiological properties and the cellular localization of HERG channels in which EGFP was tagged either to the C terminus (HERG/EGFP) or to the N terminus (EGFP/HERG). These fusion constructs were transiently expressed in human embryonic kidney (HEK) 293 cells, and the whole-cell patch-clamp configuration and a confocal laser scanning microscope with primary anti-HERG antibodies and fluorescently labeled secondary antibodies were used. For EGFP/HERG channels the deactivation kinetics were faster and the peak tail current density was reduced when compared to both wild-type HERG channels and HERG/EGFP channels. Laser scanning microscopic studies showed that both fusion proteins were localized in the cytoplasm and on discrete microdomains in the plasma membrane. The extent of labeling with anti-HERG antibodies of HEK 293 cells expressing EGFP/HERG channels was less when compared to HERG/EGFP channels. In conclusion, both electrophysiological and immunocytochemical studies showed that EGFP/HERG channels themselves have a protein trafficking defect. HERG/EGFP channels have similar properties as untagged HERG channels and, thus, might be especially useful for fluorescence microscopy studies.     

绿色荧光转基因大鼠模型的建立

目的随着干细胞研究的推进,大鼠干细胞的研究日趋迫切。本研究旨在为活体荧光影像系统、干细胞归巢、细胞移植体内示踪研究,提供绿色荧光蛋白EGFP转基因大鼠模型。方法通过显微注射方式获得EGFP转基因大鼠,采用活体荧光影像系统、激光共聚焦显微镜,对EGFP转基因大鼠各个组织的荧光表达水平进行比较;采用流式细胞术检测转基因大鼠血液和骨髓细胞、骨髓干细胞的荧光标记率,筛选骨髓干细胞高效标记绿色荧光的转基因大鼠。结果建立了心脏、肝脏、肌肉、肺、胰腺、脑、膀胱、胃、肾脏、肠和脾脏组织中,系统性表达EGFP的SD-TgN（ACT-EGFP-1）ZLFILAS转基因大鼠;流式细胞术检测表明,该品系血液细胞绿色荧光标记率为94.4%,骨髓干细胞绿色荧光标记率为97.8%。结论建立了多组织系统性高表达绿色荧光,骨髓干细胞荧光标记率高达95%以上的转基因大鼠,为影像分析,造血干细胞的归巢等研究提供了大鼠模型。     

Simultaneous analysis of the cyan, yellow and green fluorescent proteins by flow cytometry using single-laser excitation at 458 nm.

BACKGROUND: Development of spectrally distinct green fluorescent protein (GFP) variants has allowed for simultaneous flow cytometric detection of two different colored mutants expressed in a single cell. However, the dual-laser methods employed in such experiments are not widely applicable since they require a specific, expensive laser, and single-laser analysis at 488 nm exhibits considerable spectral overlap. The purpose of this work was to evaluate detection of enhanced cyan fluorescent protein (ECFP) in combination with the enhanced green (EGFP) and enhanced yellow (EYFP) fluorescent proteins by flow cytometry. METHODS: Cells transfected with expression constructs for EGFP, EYFP, or ECFP were analyzed by flow cytometry using excitation wavelengths at 458, 488, or 514 nm. Fluorescence signals were separated with a custom optical filter configuration: 525 nm shortpass and 500 nm longpass dichroics; 480/30 (ECFP), 510/20 (EGFP) and 550/30 (EYFP) bandpasses; 458 nm laser blocking filters. RESULTS: All three fluorescent proteins when expressed individually or in combination in living cells were excited by the 458 nm laser line and their corresponding signals could be electronically compensated in real time. CONCLUSIONS: This method demonstrates the detection of three fluorescent proteins expressed simultaneously in living cells using single laser excitation and is applicable for use on flow cytometers equipped with a tunable argon ion laser.     

鸡β-肌动蛋白第一内含子对EIAV转移载体外源蛋白表达能力的影响

以马传染性贫血病毒（equine infectious anemia virus, EIAV）基因转移载体pcPPTWPRE为基础,用含不同长度片段的鸡β-肌动蛋白启动子替换原有的人巨细胞病毒立即早期启动子（CMVIEp）启动外源基因的表达,分别构建了2个基因转移载体,含部分第一内含子的pWCAGP0.8和含全长内含子的pWCAGP1.6。连同在其它研究中构建的不含内含子的质粒pcPPTWCAG（另文发表）,采用磷酸钙法分别转染HEK293细胞和DF-1细胞, 利用荧光显微镜观察报告基因eGFP蛋白的表达。并利用流式细胞仪定量。统计学分析结果表明,在HEK293细胞中,pcPPTWCAG、pWCAGP0.8和 pWCAGP1.6表达阳性率分别为47.9%、46.1%和23.8%,转染后48h,收获细胞,利用流式细胞仪比较转染细胞中EGFP表达阳性细胞的百分率。结果表明,在HEK293细胞中,pcPPTWCAG、pWCAGP0.8 和pWCAGP1.6的阳性细胞分别占计数细胞的47.9%、46.1%和23.8%;在DF-1细胞中,依次分别为12.4%、9.5%和4.2%,pcPPTWCAG与pWCAGP1.6差异显著。本研究表明不含内含子的EIAV载体质粒表达EGFP的能力高于含部分和全长内含子的载体。     

异源基因α1,3半乳糖转移酶与增强型绿色荧光蛋白融合对荧光蛋白表达的影响

目的 研究异源(猪)基因α1,3半乳糖转移酶(3GT)与增强型绿色荧光蛋白(EGFP)基因形成的融合蛋白对其荧光表达量的影响.方法 BamHI,EcoRI酶切pcDNA3.1-α1,3GT重组载体后,回收含α1,3GT的片段,与BamHI、EcoRI酶切回收的pEGFP-N1载体连接,并酶切、测序鉴定重组真核表达载体p...     

表达EGFP报告基因口蹄疫病毒亚基因组复制子的构建

【目的】构建含有EGFP报告基因的口蹄疫病毒(FMDV)亚基因组复制子系统。【方法】利用融合PCR方法,将EGFP报告基因替换O型FMDV全长c DNA克隆中的前导蛋白Lb和结构蛋白P1基因,构建含有EGFP报告基因的FMDV亚基因组复制子FMDV-EGFP。复制子质粒连续转化、测序检验复制子载体的稳定性。Not I线性化的复制子FMDV-EGFP用脂质体介导法转染表达T7 RNA聚合酶的BSR/T7细胞后,不同时间段观察EGFP荧光表达情况。转染的细胞用流式、间接免疫荧光、RT-PCR和Western blot检测该复制子载体的自主复制能力和口蹄疫病毒蛋白的表达情况。【结果】复制子质粒的连续转化及测序表明报告基因可以稳定存在。FMDV-EGFP复制子转染BSR/T7细胞3 h后在荧光显微镜下能够看到绿色荧光,EGFP荧光信号随着转染时间的延长逐渐增加,并且荧光信号可持续6 d以上。转染24 h后的细胞流式分析显示转染的细胞中有6.0%发出荧光,说明构建的复制子载体能够有效表达EGFP蛋白。另外,间接免疫荧光、RT-PCR和Western blot方法也检测到该复制子RNA在BSR/T7细胞中能够进行自主复制,并且能够表达病毒的非结构蛋白。【结论】含有EGFP报告基因的FMDV亚基因组复制子的成功构建为进一步研究病毒复制、翻译机制及筛选抗病毒药物等奠定了坚实的基础。     

Production of transgenic embryos through nuclear transfer using ovine fetal fibroblasts transferred with foreign genes

Human ALR gene sequence was amplified by PCR from human total DNA and inserted into pIRES(2)-EGFP vector. The bicistronic eukaryotic expression vector, pIRES-EGFP/ALR, expressing EGFP, Neo(r) and ALR genes was constructed. Sheep fetal fibroblast cells (sEFCs) were transfected with pIRES-EGFP/ALR by the induction of lipofectAMINE(TM). The positive cell clones were selected with medium containing G418 (800 μg/mL). The fluorescence of transgenic cells was examined with a confocal laser scanning microscope. The expression of ALR gene was tested by PCR, RT-PCR and immuno-histochemical staining. The transgenic cells were used as donors for nuclear transfer to enucleated ovine oocytes. Transgenic embryos were tested by confocal laser scanning microscope and immuno-histochemical staining. Results showed that the EGFP and ALR genes linked with IRES were coexpressed simultaneously in sFFCs; the blastocysts formed by nuclear transfer using tranfected donor cells are all transgenic blastocysts. EGFP, ALR and Neo(r) gene were all expressed in the transgenic embryos. In conclusion that a method to construct the positive embryos before pre-implantation which stably express ALR gene by the indication of EGFP expression has been successfully established. The application of this method can simplify the procedure of testing the targets and contribute to the efficiency increasing of transgenic domestic animal production.     

ZNF268基因启动子区的克隆及功能分析

锌指基因家族是人体中最大的基因家族,它参与细胞分化、胚胎发育,并与许多疾病的发生相关。人类ZNF268基因是一个在人胚肝中特异性表达的C2H2型锌指基因,并可能在人的早期肝脏发育中起重要作用。为了研究ZNF268基因表达调控的分子机制,以正常人总基因组为模板PCR扩增了ZNF268基因的5′调控区2533bp片段,并将此片段插入启动子缺失的EGFP(增强型绿色荧光蛋白)载体构建了重组质粒pZNF268—EGFP。用脂质体介导的方法将pZNF268—EGFP转染NIH／3T3、COS7、K562、HeLa4个细胞系。在激光共聚焦显微镜下观察绿色荧光的表达,发现在每个细胞系中,转染了重组质粒pZNF268—EGFP的细胞均有荧光表达,但其起始表达时间均晚于转染了阳性对照质粒pEGFP—C1的细胞且荧光较弱。这表明ZNF268基因的5′调控区2．5kb片段是一个有功能的启动子,但该启动子与CMV启动子相比活性较弱。选择易培养的HeLa细胞系用于缺失研究。将一系列5′端—2456bp至—20bp缺失、3′端均为 77bp的缺失片段插入启动子缺失的CAT(氯酶素酰胺转移酶)载体构建了一系列重组质粒。将这些重组质粒转染HeLa细胞系进行缺失分析,并通过共转染pCMV—Sport—βgal质粒校正转染效率。结果表明,ZNF268启动子—2456～—1639bp区域可能含有正调控元件,—1244～—1013bp和—525～—156bp区域可能含有负调控元件,ZNF268启动子激活转录的一个重要区域位于—156～—20bp。