Visualization of agonist-induced association and trafficking of green fluorescent protein-tagged forms of both beta-arrestin-1 and the thyrotropin-releasing hormone receptor-1.

A fusion protein (beta-arrestin-1-green fluorescent protein (GFP)) was constructed between beta-arrestin-1 and a modified form of the green fluorescent protein from Aequorea victoria. Expression in HEK293 cells allowed immunological detection of an 82-kDa cytosolic polypeptide with antisera to both beta-arrestin-1 and GFP. Transient expression of this construct in HEK293 cells stably transfected to express the rat thyrotropin-releasing hormone receptor-1 (TRHR-1) followed by confocal microscopy allowed its visualization evenly distributed throughout the cytoplasm. Addition of thyrotropin-releasing hormone (TRH) caused a profound and rapid redistribution of beta-arrestin-1-GFP to the plasma membrane followed by internalization of beta-arrestin-1-GFP into distinct, punctate, intracellular vesicles. TRH did not alter the cellular distribution of GFP transiently transfected into these cells nor the distribution of beta-arrestin-1-GFP following expression in HEK293 cells lacking the receptor. To detect potential co-localization of the receptor and beta-arrestin-1 in response to agonist treatment, beta-arrestin-1-GFP was expressed stably in HEK293 cells. A vesicular stomatitis virus (VSV)-tagged TRHR-1 was then introduced transiently. Initially, the two proteins were fully resolved. Short term exposure to TRH resulted in their plasma membrane co-localization, and sustained exposure to TRH resulted in their co-localization in punctate, intracellular vesicles. In contrast, beta-arrestin-1-GFP did not relocate or adopt a punctate appearance in cells that did not express VSV-TRHR-1. Reciprocal experiments were performed, with equivalent results, following transient expression of beta-arrestin-1 into cells stably expressing VSVTRHR-1-GFP. These results demonstrate the capacity of beta-arrestin-1-GFP to interact with the rat TRHR-1 and directly visualizes their recruitment from cytoplasm and plasma membrane respectively into overlapping, intracellular vesicles in an agonist-dependent manner.     

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。     

Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis.

Thyrotropin-releasing hormone receptor (TRHR) has already been cloned in mammals wherethyrotropin-releasing hormone (TRH) is known to act as a powerful stimulator of thyroid-stimulating hormone (TSH) secretion. The TRH receptor of amphibians has not yet been characterized, although TRH is specifically important in the adaptation of skin color to environmental changes via the secretion of alpha-melanocyte-stimulating hormone (alpha-MSH). Using a dege-nerate PCR strategy, we report on the isolation of three distinct cDNA species encoding TRHR from the brain of Xenopus laevis. We have designated these as xTRHR1, xTRHR2 and xTRHR3. Analysis of the predicted amino acid sequences revealed that the three Xenopus TRHRs are only 54-62% identical and contain all the highly conserved residues constituting the TRH binding pocket. Amino acid sequences and phylogenetic analysis revealed that xTRHR1 is a member of TRHR subfamily 1 and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype awaiting discovery in other animal species. The three Xeno-pus TRHRs have distinct patterns of expression. xTRHR3 was abundant in the brain and much scarcer in the peripheral tissues, whereas xTRHR1 was found mainly in the stomach and xTRHR2 in the heart. The Xenopus TRHR subtype 1 was found specifically in the intestine, lung and urinary bladder. These observations suggest that the three xTRHRs each have specific functions that remain to be elucidated. Expression in Xenopus oocytes and HEK-293 cells indicates that the three Xenopus TRHRs are fully functional and are coupled to the inositol phosphate/calcium pathway. Interestingly, activation of xTRHR3 required larger concentrations of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation.     

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。     

Thyrotropin-releasing hormone receptor processing: role of ubiquitination and proteasomal degradation

These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.     

ω-3多聚不饱和脂肪酸脱氢酶的真核表达及鉴定

目的：构建ω-3多聚不饱和脂肪酸脱氢酶真核表达载体,并在293T细胞（人胚肾细胞）中实现表达。方法：通过RT-PCR法扩增得到ω-3多聚不饱和脂肪酸脱氢酶基因fat1,构建重组真核表达载体pCMV－Myc－fat1,用脂质体法转染293T细胞,Western Blot检测fat1的表达,并用间接免疫荧光（IFA）确定其在293T细胞中的定位情况。结果：构建真核表达质粒pCMV－Myc－fat1,转染293T细胞后,可检测到细胞内有fat1的表达并确定其在细胞中的位置。结论：成功构建真核表达质粒pCMV－Myc－fat1,可检测出细胞内有fat1的表达并确定其在细胞膜和细胞质内均有表达,为进行fat1的功能研究奠定了基础。     

比格犬ERβ1293基因真核载体的构建及其在HEK293T细胞中的表达和定位

目的 观察比格犬Myc 标签ERβ1293重组真核表达载体在HEK293T细胞中的表达及定位.方法 以pEGFP-N1-ERβ1293重组真核表达载体为模板,PCR保真酶扩增得到ERβ1293基因编码区全长.Myc标签ERβ1293重组真核载体瞬时转染HEK293T细胞,运用蛋白质印迹技术（Western blotting）和间接免疫荧光技术（indirect immunofluorescence,IF）鉴定pcDNA3.1-Myc-ERβ1293在HEK293T细胞中的表达和定位情况.结果 成功构建pcDNA3.1-Myc-ERβ1293重组真核表达载体,转染至HEK293T细胞中.Western blotting检测有蛋白条带表达,共聚焦显微镜下观察IF处理后的转染细胞,荧光定位于细胞质.结论 前期实验得到比格犬ERβ剪切异构体ERβ1293编码区序列,缺失第四外显子,导致其与配体结合能力减弱或消失,因此目的 基因编码蛋白定位在细胞中发生变化.     

Activation-induced subcellular redistribution of Gs alpha.

We have examined the subcellular distribution of alpha s, the alpha subunit of the heterotrimeric G protein Gs, by using immunofluorescence microscopy. In transiently transfected HEK293 cells, wild-type alpha s localizes to the plasma membrane. However, a mutationally activated alpha s (alpha sR201C) is diffusely distributed throughout the cytoplasm. Similarly, cholera toxin activation of alpha s causes it to redistribute from the plasma membrane to cytoplasm in stably transfected cells. In HEK293 cells stably transfected with alpha s and the beta 2-adrenergic receptor (beta-AR), stimulation of the beta-AR by the agonist isoproterenol also causes a translocation of alpha s from the plasma membrane to cytoplasm. Replacing the agonist with antagonist allows alpha s to return to the plasma membrane, demonstrating the reversibility of alpha s translocation. Receptor-activated alpha s does not colocalize with internalized beta-AR at endosomes. Incubation of cells in hypertonic sucrose to inhibit clathrin-coated pit-mediated endocytosis of agonist-activated beta-AR failed to block agonist-stimulated redistribution of alpha s. These findings demonstrate that activated alpha s reversibly undergoes a translocation from the plasma membrane to cytoplasm and begin to address the relationship between regulated trafficking of a seven-transmembrane receptor and its cognate G protein.     

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.     

人Lrp蛋白在细胞中的定位及LPS对其表达的影响

为了对脂多糖应答基因(lrp)的功能进行深入的研究,用镍离子螯合柱(Ni-NT)纯化后的 全长Lrp蛋白免疫新西兰大白兔制备多克隆抗体并吸附去除非特异性反应成分.Western 印迹表明,吸附纯化后的抗体可以与Lrp蛋白特异结合,并有较高免疫印迹滴度,为Lrp功能研究提供了重要的工具.激光共聚焦扫描荧光显微镜检测显示Lrp主要位于细胞核膜周围.Western印迹、RT-PCR以及细胞免疫组化染色结果都表明,用LPS刺激后,lrp在人HEK293 和U937细胞内的表达均有明显的上升.结果提示,Lrp可能与对Lrp介导的反应有关.     

SCG10基因重组质粒的构建及蛋白表达和定位

目的构建SCG10真核表达载体并证实融合蛋白在细胞内表达及定位。方法以人胎脑cDNA文库为模板,PCR扩增SCG10全长编码基因,亚克隆至pEGFP-C1表达载体中。将构建的重组质粒测序并转染到人胚肾HEK293中,提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察pEGFP-SCG10在HEK293细胞内定位。结果 SCG10全长基因序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段大小540bp。Western blot检测到了融合蛋白表达,分子量约为48kD。pEGFP-SCG10在细胞内定位以细胞浆为主,在细胞核少量表达。结论成功构建了SCG10全长基因真核表达载体,pEGFP-SCG10蛋白主要定位于HEK293细胞浆内。     

Heterodimerization, trafficking and membrane topology of the two proteins, Ost alpha and Ost beta, that constitute the organic solute and steroid transporter

Co-immunoprecipitation studies using mouse ileal proteins and transfected HEK-293 (human embryonic kidney-293) cells revealed that the two proteins, Ostalpha and Ostbeta, which generate the organic-solute transporter are able to immunoprecipitate each other, indicating a heteromeric complex. Mouse ileal Ostalpha protein appeared on Western blots largely as bands of 40 and 80 kDa, the latter band consistent with an Ostalpha homodimer, and both of these bands were sensitive to digestion by the glycosidase PNGase F (peptide:N-glycosidase F). Ostbeta appeared as bands of 17 and 19 kDa, and these bands were not sensitive to PNGase F. Both the 40 and 80 kDa forms of Ostalpha, and only the 19 kDa form of Ostbeta, were detected among the immunoprecipitated proteins, indicating that the interaction between Ostalpha and Ostbeta is associated with specific post-translational processing. Additional evidence for homodimerization of Ostalpha and for a direct interaction between Ostalpha and Ostbeta was provided by BiFC (bimolecular fluorescence complementation) analysis of HEK-293 cells transfected with Ostalpha and Ostbeta tagged with yellow-fluorescent-protein fragments. BiFC analysis and surface immunolabelling of transfected HEK-293 cells also indicated that the C-termini of both Ostalpha and Ostbeta are facing the intracellular space. The interaction between Ostalpha and Ostbeta was required not only for delivery of the proteins to the plasma membrane, but it increased their stability, as noted in transfected HEK-293 cells and in tissues from Ostalpha-deficient (Ostalpha-/-) mice. In Ostalpha-/- mice, Ostbeta mRNA levels were maintained, yet Ostbeta protein was not detectable, indicating that Ostbeta protein is not stable in the absence of Ostalpha. Overall, these findings identify the membrane topology of Ostalpha and Ostbeta, demonstrate that these proteins are present as heterodimers and/or heteromultimers, and indicate that the interaction between Ostalpha and Ostbeta increases the stability of the proteins and is required for delivery of the heteromeric complex to the plasma membrane.     

Homo- and hetero-oligomerization of thyrotropin-releasing hormone (TRH) receptor subtypes. Differential regulation of beta-arrestins 1 and 2

G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo- and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either beta-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only beta-arrestin 2 could enhance TRHR2 internalization. The preference for beta-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a beta-arrestin 2 knockout or a beta-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both beta-arrestins. The differential beta-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and beta-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with beta-arrestins, could also enhance TRHR2/beta-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the beta-arrestins and also provides the first evidence that the interactions of TRHRs with beta-arrestin may be altered by hetero-oligomer formation.     

反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的表达

目的:利用反转录病毒载体构建猪载脂蛋白B mRNA编辑酶催化多肽样蛋白(APOBEC)3F重组质粒,并实现其在猪肾细胞PK15中的表达。方法:用RT-PCR方法扩增五指山猪来源的外周血淋巴细胞APOBEC3F基因,将其定点插入反转录病毒载体pMSCV neo中,同时于插入位点两侧分别添加FLAG和GFP标签,构建重组质粒pMSCV-FLAG-A3F-GFP,并进行酶切、测序鉴定;将鉴定正确的重组质粒与pVSV-G、pGag-Pol共转染包装细胞HEK293T,分别于转染后48~72 h收集细胞的培养上清以获得假型病毒粒子;用该假型病毒感染猪源细胞PK15,通过PCR、Western印迹检测目的基因的整合及表达。结果:PCR扩增到1254 bp的猪APOBEC3F基因,重组质粒pMSCV-FLAG-A3F-GFP经酶切、测序,结果无误;3质粒共转染HEK293T细胞包装出的假型病毒感染PK15细胞后观察到GFP表达;从感染假型病毒的PK15细胞基因组中扩增到1254 bp的猪APOBEC3F基因,Western印迹检测到78.1×103的猪APOBEC3F蛋白的表达。结论:实现了反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的整合与表达,为深入研究该分子对猪内源性反转录病毒(PERV)的抑制作用奠定了基础。     

大熊猫FOXL2基因的克隆、序列分析及表达

从大熊猫基因组中克隆了FOXL2基因,并对其进行序列分析及原核表达和真核表达.将FOXL2编码区序列克隆到原核表达载体pET-32a(+)中,转化大肠杆菌BL21,经IPTG诱导表达出FOXL2重组蛋白.成功构建了真核表达载体FOXL2-pcDNA3.1/V5-His C,并通过脂质体介导转染HEK293细胞,Western blot检测FOXL2蛋白表达.SDS-PAGE分析表明,FOXL2重组蛋白在诱导4h后表达量达到峰值,其大小约为58.9 kDa,Western blot分析结果显示重组蛋白能够被抗His单克隆抗体特异性识别.FOXL2基因的克隆及其表达为进一步进行FOXL2的活性检测以及应用研究奠定了基础.     

New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.     

The glycosyl phosphatidylinositol anchor of human T-cadherin binds lipoproteins

T-cadherin (T-cad) is a Ca(2+)-dependent cell adhesion glycoprotein bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad expressed on vascular smooth muscle cells (SMC) binds lipoproteins on blot. To analyze the molecular basis for the interaction of T-cad with lipoproteins we expressed recombinant human T-cad in HEK293 cells. Whereas membrane-bound T-cad from SMC and T-cad transfected HEK293 cells bind lipoproteins, T-cadherin proteins cleaved from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) do not. The lipoprotein-binding function is also lacking both for a recombinant human T-cad expressed in HEK293 cells without the GPI signal sequence, and for a human T-cad form expressed in Escherichia coli that contains the signal sequence for GPI attachment but is not modified with a GPI. We conclude that the GPI moiety of T-cadherin is necessary and sufficient to mediate lipoprotein binding.     

Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0.142 for wild type (p = 0.073). The data obtained indicate that deleting 30 amino acids from the first intracellular loop of CFTR affects both processing and function of the CFTR chloride channel.     

肿瘤内皮标志物8同型物Ⅰ基因真核表达载体的构建及其在HEK293F细胞中的表达

目的:构建肿瘤内皮标志物8(TEM8)基因真核表达载体,实现TEM8在HEK293F细胞中的外源表达。方法:用PCR技术扩增TEM8基因,经限制性酶切、连接、转化,插入pcDNA3.1(+)-EGFP真核表达载体,并通过脂质体将TEM8表达质粒转染至HEK293F细胞中,Western印迹检测TEM8的表达。结果:PCR扩增得到TEM8基因,构建了真核表达载体pcDNA3.1(+)-TEM8-EGFP并转染HEK293F细胞,经G418加压筛选及有限稀释法得到生长性状良好、表达效率高的单克隆细胞系TEM8-EGFP/HEK293F;Western印迹证明过表达细胞系TEM8-EGFP/HEK293F显著表达TEM8蛋白。结论:构建了表达TEM8的重组HEK293F工程细胞系TEM8-EGFP/HEK293F,为进一步研究TEM8的生理功能奠定了基础。