LIM结构域蛋白FHL1C抑制Notch信号途径的转录激活研究

目的：克隆人表达FHL1C基因以及建立真核表达载体和慢病毒载体。方法：从人的骨骼肌细胞来源的cDNA中扩增并克隆人FHL1C基因的编码区,连接至pMD-18T载体酶切鉴定后测序。序列测定确认后,双酶切pMD18T-FHL1C回收片段,插入真核表达载体,构建真核表达载体pCMV-Myc-FHL1C酶切鉴定正确后,转染Hela细胞及Cos7细胞,用Western Blot检测其在转染细胞中的表达,用双荧光素报告基因系统检测其对Notch信号作用。构建FHL1C-IRES-GFP表达单元,建立慢病毒表达载体plen-ti6/V5-FHL1C-IRES-GFP,包装慢病毒后感染培养的细胞,用免疫荧光显微镜、Western Blot检测分析其在被感染的细胞中的表达。结果：通过PCR方法成功扩增了人FHL1C基因的编码区。通过转染Hela及Cos7细胞,使用Western Blot检测其蛋白水平表达,双荧光报告基因系统分析均能够下调激活的Notch信号。成功构建了FHL1C慢病毒表达载体pLenti6/V5-FHL1C-IRES-GFP,包装慢病毒,把获取的慢病毒感染细胞后通过荧光显微镜证实被感染的细胞绿色荧光蛋白正确表达,Western Blot检测证实其表达。结论：成功建立起FHL1C真核表达载体及慢病毒表达系统,为研究急性T淋巴细胞白血病与Notch信号转导通路之间的关系奠定了基础。     

含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体的制备与表达分析

目的:制备含分泌型萤光素酶和绿色荧光蛋白双报告基因的慢病毒载体,为慢病毒载体的进一步广泛应用奠定基础。方法:克隆构建含分泌型萤光素酶和绿色荧光蛋白双报告基因的转基因载体pCS-gluc-2A-eGFP,酶切与序列分析鉴定其正确后,与包装质粒pCMVHR’Δ8.2、包膜质粒pVSV-G共转染293FT细胞,获得含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体;重组慢病毒载体感染A549、Huh7细胞后,用荧光显微镜直接观察报告基因GFP的表达,或取细胞上清实时检测分泌型萤光素酶的表达。结果:制备了含双报告基因的重组慢病毒载体,感染细胞后可以活体观察绿色荧光蛋白的表达,也可以快速灵敏地检测到分泌型萤光素酶的表达。结论:所获含分泌型萤光素酶和绿色荧光蛋白双报告基因的重组慢病毒载体感染效率高,表达易于活体实时检测,灵敏度高。本研究为慢病毒载体的广泛应用奠定了基础。     

真核表达载体pIRES2-EGFP-Nurr1的构建及鉴定

目的:构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因Nurr1的真核表达载体pIRES2-EGFP-Nurr1,并检测其在293T细胞中的表达。方法:采用反转录-聚合酶链式反应(RT-PCR)方法从大鼠黑质中获取Nurrl基因,连接T载体测序正确后与真核空载体pIRES2-EGFP一起,经Nhe1和Xho1双酶切,T4 DNA连接酶连接,构建pIRES2-EGFP-Nurr1;真核表达载体pIRES2-EGFP-Nurr1测序正确后采用脂质体法将其转染293T细胞,倒置荧光显微镜下观察转染效率,PCR检测Nurr1基因mRNA水平的表达情况,免疫印迹试验(Western Blot)检测Nurr1蛋白的表达水平。结果:酶切及测序鉴定证实成功构建了重组真核表达载体pIRES2-EGFP-Nurr1;293T细胞转染pIRES2-EGFP-Nurr1后可以高度表达绿色荧光,有效转录Nurr1基因并正确的高表达Nurr1蛋白。结论:成功构建Nurr1真核表达载体且在293T细胞中高水平表达,为进一步转染大鼠骨髓间充质干细胞(BMSCs),基因治疗帕金森病奠定基础。     

在表达HCV Core蛋白的肝癌细胞中建立Notch1低表达稳定转染细胞株

目的筛选并包装Notch1基因shRNA低表达的慢病毒,感染稳定表达丙型肝炎病毒(hepatitis C virus,HCV)核心蛋白(core)的肝癌细胞,在此基础上建立低表达Notch1基因的SMMC7721-Core稳定转染细胞株。方法设计针对Notch1基因mRNA的干扰序列,并将其连接入载体质粒,转染HEK293T细胞,构建带有Notch1基因的慢病毒表达载体,感染稳定表达HCV Core蛋白的人肝癌细胞株SMMC7721-Core,采用定量聚合酶链反应(quantitative PCR,qPCR)和免疫印迹法(Western blot)检测干扰效果。结果构建了带有干扰Notch1基因的慢病毒表达载体,感染SMMC7721-Core细胞后筛选获得稳转细胞株,qPCR测得Notch1 mRNA低表达,Western blot检测到Notch1蛋白低表达。结论成功构建了Notch1低表达SMMC7721-Core人肝癌稳定转染细胞株,为进一步开展对HCV Core蛋白诱导肝癌过程中Notch1所起作用的研究提供了依据。     

小鼠EVL 基因的克隆和真核表达载体的构建

目的:构建小鼠EVL(Ena/VASP like)基因的真核表达载体,为深入研究EVL的功能奠定基础.方法:采用PCR方法,从小鼠cDNA文库中,扩增出1245bp的EVL编码区片段,经电泳、胶回收后连接入pMD- 18T载体中,测序鉴定正确.用BamHI和HincⅡ双酶切,定向克隆EVL编码区片段到真核表达载体pcDNA3.1中,用限制性内切酶酶切鉴定重组质粒正确后.将重组质粒转染入HELA细胞中,以RT-PCR检测EVL的mRNA的表达,以Western Blot检测EVL蛋白的表达.结果:酶切鉴定结果显示小鼠EVL编码区基因被成功克隆入真核表达载体pcDNA3.1中;RT-PCR和Western Blot结果以及免疫荧光染色显示Hela细胞中有EVL的mRNA和蛋白的表达.结论:成功获得pcDNA3.1 -EVL的真核表达载体,为进一步深入研究EVL蛋白的功能奠定了基础.     

沙眼衣原体真核表达质粒pcDNA4/CT703的构建及其表达

目的：构建含沙眼衣原体（Chlamydia trachomatis, Ct）基因CT703的真核重组表达质粒pcDNA4/CT703,并检测其在HeLa细胞中的表达.方法：利用RT-PCR扩增CT703基因,然后将其亚克隆到真核表达载体pcDNA4,PCR、双酶切和测序检测重组质粒.将正确的重组质粒瞬时转染HeLa细胞,免疫荧光和Western Blot实验检测重组质粒目的蛋白表达. 结果：经PCR、双酶切和测序鉴定后,成功构建了真核重组表达质粒pcDNA4/CT703,将其转染HeLa细胞后,免疫荧光和Western Blot实验能检测到目的蛋白的表达.结论：成功构建了重组质粒pcDNA4/CT703,并能在HeLa细胞中表达,为进一步研究CT703的功能奠定了基础.     

小鼠pEGFP-Hoxa11真核表达载体的构建及表达

目的 构建和鉴定Hoxa11和EGFP双基因共表达真核载体.方法 采用DNA重组技术,将目的 基因Hoxa11克隆至含有报告基因EGFP的pEGFP-N1真核表达载体中,构建的真核表达载体pEGFP-Hoxa11经PCR,双酶切及基因测序鉴定;转染至CHO细胞,荧光显微镜下观察重组质粒的表达,提取细胞蛋白Western印迹检测蛋白表达.结果 pEGFP-Hoxa11重组质粒构建成功.构建的真核表达载体pEGFP-Hoxa11能在CHO细胞中有效表达.结论 成功构建了共表达Hoxa11和EGFP的真核表达载体,并能在CHO细胞中有效表达.为进一步研究Hoxa11的功能提供实验基础.     

Netrin-1慢病毒表达载体的构建及鉴定

目的:构建携带有Netrin-1基因的逆转录病毒载体,为研究Netrin-1在神经发育中的作用奠定基础。方法:PCR扩增Netrin-1基因片段后,将其克隆入慢病毒表达载体pLXSN;通过PCR、酶切、测序鉴定重组质粒。重组质粒转染PA317包装细胞后获得包装的病毒颗粒。病毒颗粒感染人脑胶质瘤细胞SW038-C2,经Western blot证明重组病毒在真核细胞内表达Netrin-1的情况。结果:经PCR扩增、酶切和测序验证,重组质粒构建正确,命名为pLX-NT。Western blot证明在感染细胞泳道有一特异性条带。结论:成功构建了能表达Netrin-1的慢病毒载体。     

人GNAS1基因克隆、真核表达载体的构建及瞬时转染Hela细胞

目的:克隆人GNAS1基因编码区序列、构建真核表达载体并瞬时转染Hela细胞。方法:通过RT-PCR克隆GNAS1基因编码序列,亚克隆至pMD19载体,测序鉴定正确后,通过酶切连接至真核表达载体pEGFP-N1,通过菌液PCR和酶切鉴定获得pEGFP-GNAS1重组质粒;表达载体以Lipofectamine2000介导转染Hela细胞,并用实时定量PCR检测外源基因在Hela细胞中的表达。结果:克隆到GNAS1基因并获得了pEGFP-GNAS1表达载体,在Hela细胞中获得了GNAS1的过量表达。结论:成功克隆了GNAS1基因编码序列,并构建其真核表达载体,瞬时转染至Hela细胞,为进一步深入研究Gsα蛋白生物学作用奠定基础。     

B19病毒XA株VP1独特区真核表达系统的建立及鉴定

目的:克隆B19病毒XA株VP1u基因,构建真核重组表达载体.方法:从已构建好的B19病毒XA株原核表达载体中获得VP1u基因,将其克隆入真核表达载体plRES2-EGFP中,经酶切鉴定并测序验证后,获得真核表达载体plRES2-EGFP-VP1u.将其转染至HeLa细胞,提取细胞总蛋白,用Western blot技术检测VP1u蛋白的表达.结果:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u,荧光显微镜下可见pIRES2-EGFP-VP1u转染HeLa细胞后表达EGFP蛋白而发出绿色荧光,Western blot证明VP1u蛋白在HeLa细胞中表达.结论:成功构建了携带人B19病毒VP1u基因的真核表达载体plRES2-EGFP-VP1u并在HeLa细胞中正确表达,为今后B19病毒VP1u基因疫苗的研究奠定基础.     

Prokaryotic expression,purification and functional characterization of human FHL3

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42°C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.     

Expression of FHL1 in gastric cancer tissue and its correlation with the invasion and metastasis of gastric cancer

This study was performed to analyze the expression of four and a half LIM domains 1 (FHL1) in gastric carcinoma tissue and its correlation with the clinicopathological characteristics of gastric cancer. In addition, the role of FHL1 in the invasion and metastasis of gastric cancer cells was investigated to provide an experimental basis for future treatments of gastric cancer. FHL1 mRNA and protein expression in gastric carcinoma and the adjacent normal gastric mucosa tissue were determined using RT-PCR and western blots. Correlations of FHL1 expression with the incidence, progression, and clinicopathological characteristics of gastric cancer were analyzed. Changes in the invasion and metastatic potential of MKN45 human gastric cancer cells were observed after the transient transfection with an eukaryotic expression vector containing full-length FHL1. Expression of FHL1 mRNA in gastric carcinoma tissue was significantly lower than that in the adjacent normal tissue (P < 0.05). FHL1 expression in gastric carcinoma tissue from patients who were positive for lymph node metastasis was significantly lower than those in patients who were negative for lymph node metastasis (P < 0.05). Lower FHL1 expression was correlated with lower degrees of differentiation, higher TNM stages, and greater invasive potential of the gastric cancer (P < 0.05). The FHL1 mRNA and protein expression patterns were similar in gastric cancer. FHL1 protein expression in gastric carcinoma tissue was significantly lower than that in the surrounding normal tissue (P < 0.05). FHL1 protein expression was significantly lower in gastric carcinoma tissue from patients who were positive for lymph node metastasis than that detected in patients with no lymph node metastasis (P < 0.05). Lower FHL1 protein expression was correlated with lower degrees of differentiation, higher TNM stages, and greater invasive potential in gastric cancer (P < 0.05). However, the expression of FHL1 was independent of the patient's gender, age, and tumor size (P > 0.05). Overexpression of FHL1 in the MKN45 human gastric cancer cell line using an eukaryotic expression vector resulted in a significant reduction in the invasiveness and metastatic ability of these cells as determined using the Transwell chamber invasion assay (P < 0.05). The decrease in or loss of FHL1 expression may be related to the incidence, progression, invasiveness, and metastatic potential of gastric cancer.     

FHL2参与调控MCF-7细胞周期

目的：研究FHL2对细胞周期阻滞的影响。方法：应用pSR-GFP/Neo载体构建FHL2-siRNA干扰载体,转染MCF-7细胞,G418筛选稳定细胞系,通过流式细胞仪检测离子辐射后细胞周期的变化。结果：利用设计的FHL2-siRNA干扰载体能够干扰细胞中FHL2的表达;当离子辐射后,FHL2敲除细胞G2/M期阻滞的程度比野生型细胞显著降低。结论：FHL2参与调控MCF-7细胞离子辐射后细胞周期G2/M期阻滞。     

FHL2转录激活结构域的定位

LIM蛋白家族成员FHL2 (fourandhalfLIMdomainprotein)在转录调节、细胞凋亡及肿瘤的发生发展中都起着重要作用。利用GAL4转录因子中的DNA结合结构域 (DBD)和含有与DBD结合序列的荧光素酶报告基因(GAL4 LUC)构建了哺乳动物细胞转录激活系统 ,并利用该系统定位了FHL2的转录激活结构域。首先将GAL4 DBD序列以正确读框插入到pcDNA3载体的多克隆位点中 ,构建成真核表达载体pDBD ,再将野生型FHL2及其不同片段以正确读框与pDBD中GAL4 DBD序列融合 ,构建成野生型FHL2及其缺失突变体表达载体。将这些表达载体分别瞬时转染 2 93T胚胎肾细胞 ,野生型FHL2及其缺失突变体都得到了表达。利用GAL4 荧光素酶报告基因对野生型FHL2及其不同突变体的转录激活活性检测表明 ,在 2 93T胚胎肾细胞和乳腺癌MCF 7细胞中 ,野生型FHL2具有转录激活活性 ,缺失N端半个LIM结构域使FHL2转录激活活性降低 ,缺失C末端第二个LIM结构域对FHL2的转录激活功能影响不大 ,缺失C末端最后一个LIM结构域则使FHL2的转录激活功能完全丧失 ,而C末端缺失 2个LIM结构域使FHL2转录激活活性又有所恢复。这说明FHL2C末端最后一个LIM结构域对其转录激活功能是必需的 ,而C末端第二个LIM结构域可能对FHL2的转录激活功能有负调控作用 ,这种负调控作用取决于     

Four and a half LIM protein 1C (FHL1C): a binding partner for voltage-gated potassium channel K(v1.5)

Four-and-a-half LIM domain protein 1 isoform A (FHL1A) is predominantly expressed in skeletal and cardiac muscle. Mutations in the FHL1 gene are causative for several types of hereditary myopathies including X-linked myopathy with postural muscle atrophy (XMPMA). We here studied myoblasts from XMPMA patients. We found that functional FHL1A protein is completely absent in patient myoblasts. In parallel, expression of FHL1C is either unaffected or increased. Furthermore, a decreased proliferation rate of XMPMA myoblasts compared to controls was observed but an increased number of XMPMA myoblasts was found in the G(0)/G(1) phase. Furthermore, low expression of K(v1.5), a voltage-gated potassium channel known to alter myoblast proliferation during the G(1) phase and to control repolarization of action potential, was detected. In order to substantiate a possible relation between K(v1.5) and FHL1C, a pull-down assay was performed. A physical and direct interaction of both proteins was observed in vitro. In addition, confocal microscopy revealed substantial colocalization of FHL1C and K(v1.5) within atrial cells, supporting a possible interaction between both proteins in vivo. Two-electrode voltage clamp experiments demonstrated that coexpression of K(v1.5) with FHL1C in Xenopus laevis oocytes markedly reduced K(+) currents when compared to oocytes expressing K(v1.5) only. We here present the first evidence on a biological relevance of FHL1C.     

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。     

The LIM proteins FHL1 and FHL3 are expressed differently in skeletal muscle

We have determined the complete mRNA sequence of FHL3 (formerly SLIM2). We have confirmed that it is a member of the family of LIM proteins that share a similar secondary protein structure, renamed as Four-and-a-Half-LIM domain (or FHL) proteins in accordance with this structure. The "half-LIM" domain is a single zinc finger domain that may represent a subfamily of LIM domains and defines this particular family of LIM proteins. The distribution of FHL mRNA expression within a variety of murine tissues is complex. Both FHL1 and FHL3 were expressed in a number of skeletal muscles while FHL2 was expressed at high levels in cardiac muscle. Localisation of FHL3 to human chromosome 1 placed this gene in the proximity of, but not overlapping with, alleles associated with muscle diseases. FHL1 and FHL3 mRNAs were reciprocally expressed in the murine C2C12 skeletal muscle cell line and this suggested that the pattern of expression was linked to key events in myogenesis.