细胞内物质转运调节分子ARFGAP3的克隆表达及生化活性

ADP核糖基化因子-GTP酶活化蛋白(ARF GAP)是重要的细胞内物质转运调节分子.在22周孕龄人胎肝cDNA文库中发现一种新基因,其编码的氨基酸序列与大鼠ARF1 GAP有32%同源性.将这种新基因命名为“ARFGAP3”,对其进行功能研究,利用逆转录-聚合酶链式反应(RT-PCR),从人胎盘总RNA中扩增ARFGAP3全长cDNA序列,并将其亚克隆到pGEM-T载体;采用RNA印迹法和斑点杂交法,检测其组织表达谱,发现在多种腺体和睾丸中有很高水平ARFGAP3基因转录,并且只有一种约2.7 kb的转录本.利用基因重组技术,构建表达质粒pBAD/Thio-ARFGAP3,在大肠杆菌中表达,采用亲和层析法纯化表达产物,利用肠激酶切除重组融合蛋白N端引导序列.检测重组ARFGAP3的生化活性,证实ARFGAP3对ARF1具有GAP活性,促进ARF1结合的GTP水解为GDP,磷脂酰肌醇二磷酸(PIP2)增强其GAP活性,而磷脂酰胆碱(PC)抑制其GAP活性.     

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.     

基因枪介导真核表达质粒转染体外培养的COS-7细胞系的实验研究

目的：建立基因枪子弹制备及转染体外培养COS-7细胞系的方法。方法：以亚精氨、氯化钙沉淀法制备子弹（DNA＋金颗粒）,利用原子力显微镜观察子弹制备情况;采用基因枪方法分别将真核表达质粒pVax-Dsred-IRES-EGFP转染对照组和实验组COS-7细胞,转染后24h,利用激光扫描共聚焦显微镜观察细胞中红、绿荧光蛋白的表达。结果：制备了基因枪子弹,DNA紧密包裹在金颗粒周围;基因枪介导的pVax-Dsred-IRES-EGFP被转染入体外培养的COS-7细胞,转染后24h可检测到红、绿荧光,而对照组则没有荧光蛋白的表达。结论：国产新芝SJ-500型基因枪能够有效介导外源基因转移,基因枪转染的COS-7细胞能够有效表达报告基因。     

一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功     

人Nephrin—GFP融合蛋白真核表达载体的构建和鉴定

目的构建人19号染色体长臂Nephrin基因和绿色荧光蛋白（green fluorescence protein,GFP）真核表达质粒,为进一步研究肾小球裂隙隔膜分子复合物构成与功能提供基础。方法设计引物,扩增真核表达质粒pEGFPN3中的GFP基因片段,将其插入nephrin原核表达载体pcDNA3．1 nephrin V5-His,重组质粒经酶切鉴定后测序,并转染至COS-7细胞观察表达情况及生物学特性。结果成功构建pcDNA3．Inephrin—GFP重组质粒,并将Nephtin—GFP融合蛋白成功表达于COS-7细胞,进一步经交联实验证明Nephtin—GFP融合蛋白具有正确的细胞膜表达。结论利用pEGFPN3和pcDNA3．1nephtinV5-His可成功重组Nephrin—GFP表达质粒,为进一步研究肾小球裂隙隔膜分子复合物构成及其功能提供有利工具。     

糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达.分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243 bp GPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒.经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况.经酶切和测序鉴定证实,所克隆的CD24 GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光.成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础.     

结核分枝杆菌Rv3586结构域基因真核表达载体的构建及鉴定

目的:构建结核分枝杆菌(Mycobacterium tuberculosis,Mtb)环二腺苷酸(cyclic diadenosine monophosphate,c-di-AMP)合成酶Rv3586结构域基因的真核表达载体,并在COS-7细胞中表达。方法:以Mtb基因组为模板PCR扩增Rv3586三个结构域基因,分别克隆入pEGFP-N3真核表达质粒,用菌落PCR、质粒酶切和测序方法对插入序列进行鉴定。通过脂质体将重组质粒转染COS-7细胞,间接免疫荧光法检测目的基因在COS-7细胞内的表达。结果:PCR成功扩增出Rv3586三个结构域基因,菌落PCR、质粒酶切和质粒测序鉴定结果表明成功插入目的片段,包含Rv3586的三个结构域基因的真核表达载体构建成功。间接免疫荧光法结果显示,Rv3586三个结构域蛋白在COS-7细胞中表达成功。结论:成功构建Rv3586三个结构域基因的真核表达载体,并在COS-7细胞中表达成功,为后续Mtb Rv3586结构域的功能和应用研究奠定了基础。     

BALB/c小鼠I-Ad αβ链RFP融合双顺反子表达载体的构建及其在COS-7中的表达

构建了在β链C端融合红色荧光蛋白(RFP)标签的BALB/c小鼠I-Adαβ链真核双顺反子表达载体pRed-IRES-I-Ad,使用LipofectAMINE2000转染COS-7细胞,用激光共聚焦显微镜观察外源蛋白在细胞中的表达与定位.I-Adαβ分子在COS-7细胞中能够以较高的效率表达,并且在COS细胞中能形成聚集状态.与通常的真核翻译帽子结构起始相比,IRES启动真核翻译系统的效率低于前者;与空质粒对比,IRES介导的真核翻译起始,RFP的表达量较低.     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的:构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法:利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果:成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论:成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的：构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法：利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果：成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论：成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

Functional characterization of novel human ARFGAP3

ADP ribosylation factors (ARFs) are critical in the vesicular trafficking pathway. ARF activity is controlled by GTPase-activating proteins (GAPs). We have identified recently a novel tentative ARF GAP derived from human fetal liver, ARFGAP3 (originally named as ARFGAP1). In the present study, we demonstrated that ARFGAP3 had GAP activity in vitro and remarked that the GAP activity of ARFGAP3 was regulated by phospholipids, i.e. phosphatidylinositol 4,5-diphosphate as agonist and phosphatidylcholine as antagonist. ARFGAP3 is a predominantly cytosolic protein, and concentrated in the perinuclear region. Its transient ectopic overexpression in cultured mammalian cells reduced the constitutive secretion of secreted alkaline phosphatase, indicating that ectopic overexpression of ARFGAP3 inhibits the early secretory pathway of proteins in vivo. These results demonstrated that ARFGAP3 is a novel GAP for ARF1 and might be involved in intracellular traffic of proteins and vesicular transport as predicted.     

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...     

小鼠天然可溶性VEGF受体基因sflt-1的克隆及鉴定

 可溶性血管内皮细胞生长因子受体 (sFlt 1 )与膜表面受体Flt 1竞争结合血管内皮细胞生长因子 (VEGF) ,并且与膜表面受体Flt 1及KDR形成异源二聚体 .完全阻断VEGF的生物学活性 ,除与sFlt 1的结合部位结构域有关外 ,还与整个蛋白质分子的高效分泌表达有关 .而蛋白质分子的高效分泌表达与蛋白质在细胞内高尔基体及内质网的加工密切相关 ,基因工程重组可溶性受体由于一些尚未明了的原因 ,往往不能高效表达 ,从而大大影响了其应用价值 .利用RT PCR技术从胚胎小鼠中扩增出天然可溶性VEGF受体基因sflt 1 ,克隆于pcDNA3载体中 ,在COS 7细胞中短暂表达 ;并克隆至pET42b载体中 ,经IPTG诱导后 ,可大量稳定表达与His Tag形成的融合蛋白 ,经HisNi柱纯化 ,可特异性结合VEGF .可溶性受体sFlt 1在肿瘤组织的高效表达可有效阻断新生血管的形成 ,从而为肿瘤的治疗探索一种方法 .     

67kD层粘连蛋白受体的克隆、表达及其对肝癌细胞侵袭能力的影响

探讨细胞膜表面 6 7kD层粘连蛋白受体 (6 7kDlamininreceptor ,6 7LR)在肝癌细胞侵袭转移中的作用 ,从肝癌细胞中提取RNA ,通过RT PCR扩增 6 7LR的前体——— 37kD层粘连蛋白受体前体(37kDlamininreceptorprecursor,37LRP)基因并定向克隆到真核表达载体pcDNA3.1 myc His(- )A ,采用脂质体将重组质粒转染到HepG2肝癌细胞中 ,通过G4 1 8筛选和RT PCR、流式细胞术鉴定 ,获得了细胞膜表面 6 7LR高表达 (阳性率为 6 9.2 % )和低表达 (阳性率为 1 1 .7% )的细胞克隆 ,采用体外细胞侵袭实验测定不同细胞的侵袭能力 ,发现膜表面 6 7LR高表达的细胞侵袭能力明显高于低表达及不表达细胞 ,说明 6 7LR在肝癌细胞侵袭转移过程中可能具有重要意义     

人TIMP-1 cDNA克隆、真核表达载体构建及在COS-7细胞中的表达

根据 Gen Bank中 TIMP- 1基因的碱基序列 ,用 RT- PCR方法从人的正常肾组织中克隆出包含信号肽在内的 TIMP- 1全长 c DNA序列 .采用 T- A克隆的方法将之插入 p CRR2 .1中间载体 ,DNA测序证实该片段序列与文献报告的完全一致 .利用亚克隆的方法将 TIMP- 1 c DNA片段克隆到 pc DNA3载体上 ,构建出 pc DNA3/ TIMP- 1的真核表达载体 ,通过脂质体 DOTAP转染至 COS-7细胞 ,Northern印迹及原位杂交证实在 COS- 7细胞上获得人 TIMP- 1的高效表达 ,细胞增殖实验表明 TIMP- 1的高产表达可促进 COS- 7细胞的增殖 ,证实了所转染人 TIMP- 1的生物活性     

The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1

Background

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.

Principal Findings

As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.

Conclusions

ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.     

Characterization, chromosomal assignment, and tissue expression of a novel human gene belonging to the ARF GAP family

We have identified and characterized a novel human ADP-ribosylation factor GTPase-activating protein (ARFGAP1) gene that is related to other members of the ARF GAP family. The full-length cDNA for human ARFGAP1 was cloned following the identification of an EST obtained by large-scale cDNA library sequencing through a Blast search of public databases. Structurally, ARFGAP1 encodes a polypeptide of 516 amino acids, which contained a typical GATA-1-type zinc finger motif (CXXCX(16)CXXC) with the four cysteine residues that are highly conserved among other members of the ARF GAP family. The conserved ARF GAP domain may emphasize the biological importance of this gene. The ARFGAP1 gene, which contained 16 exons ranging from 0.5 to 9.3 kb, was mapped to human chromosome 22q13.2-q13.3 using radiation hybridization and in silico analyses. ARFGAP1 is strongly expressed in endocrine glands and testis. Interestingly, the expression of ARFGAP1 in testis is about sixfold higher than that in ovary, indicating a possible role of ARFGAP1 in the physiological function of sperm. Expression of ARFGAP1 in four human fetal tissues and seven cancer cell lines was also detected.