羟胺切割Neurturin融合蛋白表达载体的构建、表达产物纯化及生物活性

 Neurturin (NTN)是新近发现的一种神经营养因子 ,是 GDNF家族的成员之一 .将 5′端引入了羟胺切割位点的 h NTN基因克隆到硫氧还蛋白融合表达载体 p Thio His A,在宿主菌 BL2 1中获得了稳定、高效表达 ,表达产物以包涵体形式存在 .在变性条件下经羟胺切割、柱层析纯化后复性 ,获得纯度达 90 %以上的 rh NTN.经鸡胚背根神经节 (DRG)培养法测定具有生物学活性 .     

高效可溶性重组蛋白表达载体的构建

本研究构建了两种高效表达可溶性重组蛋白的原核表达载体。一种载体由HisSUMO序列与pET30a(+)载体连接而成(命名为HisSUMO Express),表达的融合蛋白用Ni-NTA纯化,用SUMO蛋白酶I切割后可获得不留任何残基的重组蛋白。SUMO-蛋白酶I价格较贵,为减少表达蛋白的成本,第二种载体即在His-SUMO和目的序列之间加入羟胺切割位点(命名为HisSUMO Economic)。在HisSUMO Economic中表达的融合蛋白用Ni-NTA纯化,羟胺液切割后可获得仅留一个甘氨酸残基的重组蛋白。以在常规表达载体中难以表达的鼠源成纤维细胞生长因子-21(mFGF-21)为例,经葡萄糖消耗实验检测其活性,验证两种表达载体的效果。结果表明mFGF-21在两种载体中均获得了高效表达,融合蛋白占菌体总蛋白的40%以上,Ni-NTA纯化后的融合蛋白分别利用羟胺切割液和SUMO蛋白酶I切割,纯化的mFGF-21成熟蛋白回收量约为54mg/L,回收率约为6%。经两种载体表达后的mFGF-21蛋白均具有生物学活性,可促进脂肪细胞消耗葡萄糖,为进一步研究提供了基础。     

人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性

成纤维细胞生长因子FGF-21是FGF家族的一个新成员, 最近的研究发现其具有调节血糖的作用, 有望成为治疗2型糖尿病的基因药物。文章应用RT-PCR技术, 从成人肝脏中克隆人源的FGF-21成熟蛋白基因, 并将其克隆到T载体上, 经测序鉴定后, 将其亚克隆到原核表达载体pSUMO上, 转入大肠杆菌Rosetta(DE3)中。鉴定阳性克隆后, 用IPTG诱导FGF-21表达, 并用Ni-NTA柱进行亲和层析纯化。以3T3-L1脂肪细胞的葡萄糖吸收实验来鉴定FGF-21表达产物促进糖吸收的活性。结果表明, FGF-21成熟蛋白基因大小为546 bp, 测序结果与GenBank数据库中的序列一致。SDS-PAGE与Western blotting结果表明: 人源FGF-21成熟蛋白大小19.4 kDa, 经3T3-L1脂肪细胞的葡萄糖吸收实验验证其具有促进葡萄糖吸收的生物活性, 并且GLUT1是FGF-21发挥生物学作用的终末执行单位。     

人源FGF-21在脂肪细胞糖代谢中的作用

近年来研究发现,成纤维细胞生长因子(FGF)-21是一种新的代谢调节因子.为了深入研究人源FGF-21（hFGF-21）的生物活性,本实验利用SUMO高效表达载体,高效表达成熟的hFGF-21,并利用小鼠3T3-L1脂肪细胞检测hFGF-21的糖代谢活性．实验结果表明,hFGF-21可促进脂肪细胞的葡萄糖吸收,且葡萄糖吸收效率呈剂量依赖性．hFGF-21作用4 h即可促进脂肪细胞糖吸收,其活性可持续24 h以上．hFGF-21与胰岛素共同作用的葡萄糖吸收效果,明显优于它们的单独作用结果,说明hFGF-21与胰岛素发挥协同作用．脂肪细胞经hFGF-21预处理后,显著增加了胰岛素促进脂肪细胞吸收葡萄糖的效率,说明hFGF-21可以增加胰岛素的敏感性．本实验为临床应用hFGF-21治疗糖尿病,增加胰岛素敏感性提供了依据．     

成纤维细胞生长因子(FGF)-21改善胰岛素抵抗肝细胞对葡萄糖的吸收和肝糖原的合成

在体外建立胰岛素抵抗肝细胞模型,探讨在胰岛素抵抗状态下成纤维细胞生长因子(FGF)-21对模型细胞糖代谢的影响及机制．将HepG2细胞置于10^-7 mol/L 的胰岛素培养基中培养24 h,建立胰岛素抵抗细胞模型．分别用不同浓度的胰岛素和FGF-21处理模型细胞,采用葡萄糖氧化酶-过氧化物酶(GOD-POD)法检测细胞对葡萄糖的摄取情况,并检查胰岛素与FGF-21的协同作用．利用实时荧光定量PCR检测FGF-21对模型细胞葡萄糖转运蛋白1(GLUT1)mRNA表达的影响,蒽酮法检测模型细胞糖原合成量,探讨FGF-21对胰岛素抵抗细胞模型葡萄糖摄取的影响及机制．结果发现,用高浓度胰岛素处理HepG2细胞24 h后,细胞对胰岛素的敏感性显著降低,说明成功建立了胰岛素抵抗细胞模型,抵抗状态可维持48 h,未发现细胞形态学变化．FGF-21能改善胰岛素抵抗模型细胞的葡萄糖摄取,参与肝糖原的合成,并与胰岛素产生协同作用．实时荧光定量PCR结果发现,FGF-21作用模型细胞后,细胞的GLUT1 mRNA表达量显著增加,说明FGF-21促进模型细胞摄取葡萄糖的作用机制与其增加GLUT1的表达有关．     

昆明鼠肠激酶轻链的原核表达、复性与纯化

目的:构建昆明小鼠十二指肠肠激酶轻链(mEKL)大肠杆菌高效表达系统,并建立复性与纯化方法。方法:RT-PCR法扩增mEKL全长cDNA,以融合表达载体pET32a克隆于大肠杆菌BL21(DE3)和Origami(DE3),采用阴离子交换层析纯化复性表达产物,经牛肠激酶消化回收mEKL。结果:扩增得到723bp的mEKL全长cDNA,经序列分析发现,昆明鼠来源的mEKLcDNA序列的Ser131密码子中存在1个同义点突变。mEKL在2种宿主菌中的表达产物均为包涵体蛋白,经稀释复性、阴离子交换层析纯化和肠激酶切割可得高纯度mEKL。结论:成功构建昆明鼠源mEKL高效表达工程菌,并建立相应的纯化方法。     

颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达

为高效表达颗粒裂解肽G13结构域并避免G13对宿主菌的毒性, 将人工合成的编码G13的基因片段, PCR扩增后克隆于原核表达载体pThioHisA中, 构建了重组表达载体pThioHisA-G13, 将其转化于大肠杆菌BL21(DE3)中, 经IPTG诱导表达融合蛋白Trx-G13, 表达产物以包涵体的形式存在, 其表达量约占细菌总蛋白的58%。包涵体蛋白经 8 mol/L尿素溶解后, 再经CNBr切割, 阳离子交换层析, 得到纯化的重组G13结构域。琼脂糖扩散法检测表明重组G13结构域多肽具有抗菌活性。     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

乙型肝炎病毒DNA聚合酶反式调节蛋白HBVDNAPTP1的表达、纯化与初步鉴定

目的构建HBVDNAPTP1基因的原核表达载体,诱导其在大肠埃希菌中表达,并对融合蛋白进行纯化。方法利用逆转录-PCR获得乙型肝炎病毒(HBV)DNA聚合酶(Polymerase)反式调节人类新基因HBVD-NAPTP1,测序正确后插入至原核表达载体pET-32a(+)中,转化BL21(DE3)宿主菌进行诱导,并利用组氨酸亲和层析方法对融合蛋白进行纯化。结果 HBVDNAPTP1原核表达载体转化宿主菌后,经0.5 mmol/L IPTG、30℃诱导5 h获得了分子量约为31 kD的HBVDNAPTP1融合蛋白的优化表达,Western blotting证实融合蛋白的特异性。亲和层析纯化后得到较纯的HBVDNAPTP1融合蛋白,每升培养菌液中可获得2.24 mg的纯化蛋白。结论成功获得纯化的HBVDNAPTP1融合蛋白,为今后开展HBVDNAPTP1的生物学功能研究奠定了物质基础。     

鸡源抗菌肽Fowlicidin-2在大肠杆菌中的 重组表达及其生物学活性鉴定

【目的】对抗菌肽Fowlicidin-2基因进行克隆与表达,并鉴定其生物学活性。【方法】根据抗菌肽Fowlicidin-2氨基酸序列,依照大肠杆菌(E.coli)密码子的偏爱性,人工设计合成其编码基因。与质粒pET-32a连接,构建重组表达载体,转化表达宿主菌E.coliBL21(DE3),IPTG诱导表达,融合蛋白经溴化氰裂解后进行纯化,测定重组抗菌肽的抑菌活性。【结果】Fowlicidin-2融合蛋白以包涵体形式表达,经溴化氰裂解后,成功释放出Fowlicidin-2,获得的重组Fowlicidin-2对革兰氏阳性菌和革兰氏阴性菌均有明显的抑菌效果。【结论】实现了抗菌肽Fowlicidin-2的重组表达,为抗菌肽的重组量化制备提供了理论基础与技术手段。     

Shikonin stimulates glucose uptake in 3T3-L1 adipocytes via an insulin-independent tyrosine kinase pathway

Type 2 diabetes is due to defects in both insulin action and secretion. In an attempt to discover small molecules that stimulate glucose uptake, similar to insulin, a cell-based glucose uptake screening assay was performed using 3T3-L1 adipocytes. Shikonin, a substance originally isolated from the root of the Chinese plant that has been used as an ointment for wound healing, was thus identified. Shikonin stimulated glucose uptake and potentiated insulin-stimulated glucose uptake in a concentration-dependent manner in 3T3-L1 adipocytes. Stimulation of glucose uptake was also observed in rat primary adipocytes and cardiomyocytes. Like insulin, shikonin-stimulated glucose uptake was inhibited by genistein, a tyrosine kinase inhibitor, and enhanced by vanadate, a tyrosine phosphatase inhibitor. However, in contrast to insulin, shikonin-stimulated glucose uptake was not strongly inhibited by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). In vitro phosphorylation analyses revealed that shikonin did not induce tyrosine phosphorylation of the insulin receptor, but significantly induced both Thr-308 and Ser-473 phosphorylation of Akt. Our results suggest that in 3T3-L1 adipocytes, shikonin action is not mediated primarily via the insulin receptor/PI3K pathway, but rather via another distinct tyrosine kinase-dependent pathway leading to glucose uptake involving Akt phosphorylation.     

Molecular determinants of FGF-21 activity-synergy and cross-talk with PPARgamma signaling

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. The precise mechanisms whereby FGF-21 regulates metabolism remain to be determined. Here we describe the early signaling events triggered by FGF-21 treatment of 3T3-L1 adipocytes and reveal a functional interplay between FGF-21 and peroxisome proliferator-activated receptor gamma (PPARgamma) pathways that leads to a marked stimulation of glucose transport. While the early actions of FGF-21 on 3T3-L1 adipocytes involve rapid accumulation of intracellular calcium and phosphorylation of Akt, GSK-3, p70(S6K), SHP-2, MEK1/2, and Stat3, continuous treatment for 72 h induces an increase in PPARgamma protein expression. Moreover, chronic activation of the PPARgamma pathway in 3T3-L1 adipocytes with the PPARgamma agonist and anti-diabetic agent, rosiglitazone (BRL 49653), enhances FGF-21 action to induce tyrosine phosphorylation of FGF receptor-2. Strikingly, treatment of cells with FGF-21 and rosiglitazone in combination leads to a pronounced increase in expression of the GLUT1 glucose transporter and a marked synergy in stimulation of glucose transport. Together these results reveal a novel synergy between two regulators of glucose homeostasis, FGF-21 and PPARgamma, and further define FGF-21 mechanism of action.     

3T3-L1脂肪细胞膜FGF-21结合蛋白的初步鉴定

成纤维细胞生长因子（fibroblast growth factor,FGF）-21是最近发现的1个可以独立调节血糖的细胞因子,有望成为治疗2型糖尿病的备选药物．但是,FGF-21调解血糖的机理尚不十分清楚．为探讨该因子功能受体,应用偶联方法,以3T3-L1脂肪细胞为靶标,以FGF-21为诱饵,在3T3-L1脂肪细胞膜上寻找结合蛋白．结果表明,生物素标记的FGF-21可与脂肪细胞膜蛋白形成分子质量大小约300 kD以上两组复合物．竞争试验显示,非标记的FGF 21可与生物素标记的FGF-21竞争、抑制标记的FGF-21参入复合物;应用非标记FGF 21剂量越大,抑制后者参入复合物的程度越强.结果提示,该复合物是FGF-21特异性的．此外,随着生物素标记的FGF-21剂量增加,观察到的标记复合物越多;但是,当FGF-21剂量达12.5 mg/L以上时,观察到的复合物数量不再增加．实验结果提示,复合物形成与FGF-21剂量相关;FGF-21特异结合的蛋白质结合位点饱和后,复合物形成量最大．同时,采用FGF受体特异性抑制剂SU5402可特异性抑制FGF-21在3T3-L1脂肪细胞中的促进葡萄糖吸收作用,提示本实验所观察到的FGF-21 膜蛋白复合物可能就是FGF-21-FGF受体     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

The insulin sensitizing effect of homoisoflavone-enriched fraction in Liriope platyphylla Wang et Tang via PI3-kinase pathway

In the present study, we screened candidates for enhancing insulin action, using glucose uptake as an indicator, from Liriope platyphylla Wang et Tang (LPWT) extract, Liliaceae, in 3T3-L1 adipocytes. The mechanism of insulin sensitizing action in the fractions was also investigated. LPWT extract with 70% MeOH was sequentially separated with Diaion HP-20 and silica gel column chromatography. The 9:1 fraction from silica gel column chromatography increased glucose uptake with 1 ng/mL up to glucose uptake with 50 ng/mL insulin. The 9:1 fraction, determined as homoisoflavone-enriched fraction, worked as an insulin sensitizer. It increased insulin stimulated glucose uptake in 3T3-L1 adipocytes, insulin responsive cells, through increased glucose transporter 4 (GLUT4) contents in the plasma membrane. GLUT4 translocation was increased through insulin receptor substrate 1 (IRS1)-PI3 kinase-Akt signaling mechanism. Thus, homoisoflavone-enriched fraction in LPWT extract played an important role as an insulin sensitizer in adipocytes.     

Chromium and vanadate combination increases insulin-induced glucose uptake by 3T3-L1 adipocytes

Insulin activates signaling pathways in target tissues through the insulin receptor and Tyr phosphorylation of intracellular proteins. Vanadate mimics insulin and enhances its actions through inhibition of protein Tyr phosphatases. Chromium is a micronutrient that enhances insulin action to normalize blood glucose, but the mechanism is not understood. Here we show that either vanadate or chromium stimulates Tyr phosphorylation of insulin receptor in mouse 3T3-L1 adipocytes compared to insulin alone, but a combination of vanadate and chromium is not additive. Phosphorylation of MAPK or 4E-BP1 as markers for insulin signaling is stimulated by vanadate plus insulin, and chromium does not enhance the effects. Vanadate robustly activates glucose uptake by 3T3-L1 adipocytes even without added insulin and increases insulin-stimulated glucose uptake. Chromium pretreatment of adipocytes slightly enhances glucose uptake in response to insulin, but significantly increases glucose uptake above that induced by insulin plus vanadate. These data show that chromium enhances glucose uptake even when Tyr phosphorylation levels are elevated by vanadate plus insulin, suggesting separate mechanisms of action for vanadate and chromium.     

Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.     

R-alpha-lipoic acid action on cell redox status, the insulin receptor, and glucose uptake in 3T3-L1 adipocytes.

The insulin signaling pathway has been reported to mediate R-alpha-lipoic acid- (R-LA-)-stimulated glucose uptake into 3T3-L1 adipocytes and L6 myotubes. We investigated the role of the thiol antioxidant dihydrolipoic acid (DHLA) and intracellular glutathione (GSH) in R-LA-stimulated glucose transport and explored the hypothesis that R-LA could increase glucose uptake into 3T3-L1 adipocytes in an oxidant-mimetic manner. R-LA pretreatment of 3T3-L1 cells stimulated glucose transport at early time points (30 min - 6 h), whereas it inhibited glucose uptake at later time points. Analysis of the oxidized and reduced content of LA in cells and medium showed that >90% of lipoic acid present was in its oxidized form. Furthermore, all oxidized forms of LA (S-, R-, and racemic LA) stimulated glucose uptake, whereas the reduced form, dihydrolipoic acid, was ineffective. Intracellular GSH levels were not changed at the early time points (before 12 h), while longer preincubation (24 - 48 h) of cells with R-LA significantly increased intracellular GSH. Pretreatment of adipocytes with R-LA increased intracellular peroxide levels at early time points (30 min - 6 h), after which it was decreased (12 - 48 h). R-LA also increased tyrosine phosphorylation of immunoprecipitated insulin receptors from 3T3-L1 adipocytes. These results indicate that (i) 3T3-L1 adipocytes have a low capacity to reduce R-LA and the oxidized form of lipoic acid is responsible for stimulating glucose uptake, (ii) R-LA modulates glucose uptake by changing the intracellular redox status, and (iii) the insulin receptor is a potential cellular target for R-LA action.