The suppression of thymic stromal lymphopoietin expression by selenium

Thymic stromal lymphopoietin (TSLP) is a key mediator of allergic diseases such as allergic rhinitis, asthma, and atopic dermatitis. Selenium (Se) has various effects such as antioxidant, antitumor, antiulcer, and anti-inflammatory effects. However, the effect of Se on the production of TSLP has not been clarified. Thus, we investigated how Se inhibits the production of TSLP in the human mast cell line, HMC-1 cells. Se suppressed the production and mRNA expression of TSLP in HMC-1 cells. The maximal inhibition rate of TSLP production by Se (10 μM) was 59.14 ± 1.10%. In addition, Se suppressed the nuclear factor-κB luciferase activity induced by phorbol myristate acetate plus A23187. In the activated HMC-1 cells, the activation of caspase-1 was increased; whereas the activation of caspase-1 was decreased by pretreatment with Se. These results suggest that Se can be used to treat inflammatory and atopic diseases through the suppression of TSLP.     

Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration

The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.     

甲胎蛋白的原核表达及复性优化

构建甲胎蛋白的原核表达载体p ET32a-AFP,对包涵体形式表达的甲胎蛋白进行复性优化。将构建的重组质粒p ET32a-AFP转化入E.coli,IPTG诱导表达后,经亲和层析纯化获得AFP包涵体,通过对复性过程、p H、添加剂等的研究摸索,获得最佳复性条件。当采用添加0.5 mol/L L-精氨酸的一步法透析复性方法,且透析液p H值为8.5,重组人AFP包涵体蛋白起始浓度为1.0 mg/m L时,复性效率最高。该复性方法获得蛋白质具有较高的回收率且操作简便。     

Human thymic stromal lymphopoietin preferentially stimulates myeloid cells.

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.     

人热休克蛋白70的原核高效表达

将人热休克蛋白基因hsp70片段克隆到高效原核表达载体pMAL-c2X中,酶切鉴定并进行DNA测序。将该重组表达载体转化大肠杆菌DH50α,用IPTG在不同温度及时间下进行诱导表达。收集细菌,菌体裂解后进行SDS-PAGE及Western blot检测,并以凝胶薄层扫描分析表达水平。结果表明,成功地构建了含人hsp70基因的表达载体pMAL-c2X／hsp70,该载体能在大肠杆菌中表达相对分子质量为110000并具有抗原活性的融合蛋白;改变诱导温度和时间,目的蛋白表达总量及可溶性部分所占比例不同。对人hsp70基因的克隆、表达,并对其进行表达条件的优化,为研究HSP70的结构、功能与临床应用提供了必要条件。     

Increased susceptibility to autoimmune gastritis in thymic stromal lymphopoietin receptor-deficient mice

Thymic stromal lymphopoietin (TSLP), mainly produced by epithelial cells, activates a variety of cell types, including dendritic cells, mast cells, T cells, and B cells. It is involved in the pathogenesis of allergic inflammation in the lung, skin, and gastrointestinal tract. In addition, TSLP promotes Th2-type intestinal immunity against helminth infection and regulates Th1-type inflammation in a mouse model of colitis, suggesting that it plays crucial roles in intestinal immune homeostasis. Although autoimmune gastritis (AIG), mediated by inflammatory Th1 responses, develops in the gastric mucosa, it is not clear whether TSLP is involved in regulating these responses in AIG. The aim of this study was to examine the roles of TSLP in the development of AIG. Because BALB/c mice thymectomized 3 d after birth (NTx mice) develop AIG, we used this model to test the role of TSLP in the development of AIG. We found that in AIG-bearing mice, TSLP was expressed in the inflamed stomach and that the serum anti-parietal cell Ab levels in neonatal thymectomized TSLPR-deficient mice (NTx-TSLPR(-/-) mice) were significantly elevated over those in NTx-TSLPR(+/+) mice. In addition, NTx-TSLPR(-/-) mice exhibited an earlier onset of AIG than that observed in NTx-TSLPR(+/+) mice. The rapid development of AIG in NTx-TSLPR(-/-) mice resulted in more aggressive CD4(+) T cell infiltration and more severe loss of parietal and chief cells in the progression phase of AIG, accompanied by enhanced production of IL-12/23p40 and IFN-γ. Taken together, these data suggested that TSLP negatively regulates the development of AIG.     

猪链球菌IgG结合蛋白的原核表达及其与不同动物IgG的结合性

猪链球菌IgG结合蛋白(SPG)是一种可与多种动物IgG结合的细胞壁蛋白,广泛地存在于猪链球菌的各个血清型中,被认为是共同抗原。然而其在猪链球菌中的生物学意义并不清楚。本实验用PCR方法从猪链球菌1/2、1、2和9型菌株基因组中扩增SPG基因,构建pET28a-SPG重组表达载体,将其转入大肠杆菌BL21菌株。IPTG诱导表达后,重组蛋白经SDS-PAGE及Western blotting鉴定为可高效表达。镍亲和层析及分子筛两步纯化后获得纯度较高的目的蛋白。Western blotting及ELISA试验结果表明,所有纯化的目的蛋白均可与不同动物IgG结合,其中与人和猪IgG的结合能力相对较高,但不同血清型猪链球菌SPG与同种动物IgG的结合活性没有明显差异。     

Optimization of the industrial production of the recombinant precursor of human insulin

Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.     

Production of a recombinant human basic fibroblast growth factor with a collagen binding domain

Summary Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-Fl) fromEscherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [³H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [³H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type 1 collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.     

人同型融合和蛋白质分选复合体亚基的原核表达、蛋白纯化及功能验证

同型融合和蛋白质分选复合体(HOPS)由VPS11、VPS16、VPS18、VPS33、VPS39和VPS41这6种蛋白组成,能够通过膜融合机制来调节生物体内的膜泡运输。已有研究表明其可以作为融合因子来促进自噬体与溶酶体膜融合过程。为在体外确定HOPS复合体与自噬性SNARE蛋白STX17是否具有直接相互作用,首先利用PCR技术从已有质粒中扩增得到6种基因的编码序列,将其连接至pGEX 4T-1-GST或pET-His-NusA原核表达载体上,经菌落PCR初步鉴定和DNA测序无误后成功构建6种原核表达重组质粒并转化至大肠杆菌BL21(DE3);利用谷胱甘肽琼脂糖树脂与镍柱对重组蛋白进行纯化,烟草蚀纹病毒(TEV)蛋白酶酶切掉GST或His-NusA标签,得到分子量约为105 kDa的HA-VPS11蛋白、97 kDa的Flag-VPS16蛋白、108 kDa的HA-VPS18蛋白、70 kDa的Flag-VPS33蛋白、97 k Da的HA-VPS39蛋白和98 kDa的Flag-VPS41蛋白;通过体外GST pull-down技术对6种蛋白的功能进行验证,证实自噬性SNARE蛋白S...     

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6?×?His–TF–CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF–CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase–human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25?mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.     

Cutting edge: direct action of thymic stromal lymphopoietin on activated human CD4+ T cells

Thymic stromal lymphopoietin (TSLP) is a cytokine that promotes CD4(+) T cell homeostasis and contributes to allergic and inflammatory responses. TSLP can act directly on mouse CD4(+) T cells, but in humans, the available data have indicated that TSLP receptors are not expressed on CD4(+) T cells and that TSLP instead activates dendritic cells, which in turn promote the proliferation and differentiation of CD4(+) T cells. We now unexpectedly demonstrate the presence of TSLP receptors on activated human CD4(+) T cells. Strikingly, whereas freshly isolated peripheral blood human T cells show little if any response to TSLP, TCR stimulation allows a potent response to this cytokine. Moreover, TSLP increases the sensitivity of human CD4(+) T cells to low doses of IL-2, augmenting responsiveness of these cells to TCR engagement. Our results establish that human CD4(+) T cells are direct targets for TSLP.     

TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.     

IL-13 induces skin fibrosis in atopic dermatitis by thymic stromal lymphopoietin

Skin fibrotic remodeling is a major feature in human atopic dermatitis (AD). Inflammation and tissue fibrosis are common consequences of Th2 responses. Elevated IL-13 and thymic stromal lymphopoietin (TSLP) have been found in the AD skin lesions. Fibrocytes can be recruited to inflamed tissues to promote wound healing and fibrosis. Dermal transgenic expression of IL-13 causes an AD-like phenotype with fibrosis and increased TSLP. However, the role of TSLP in fibrotic remodeling is unknown. In this study, we investigated the role of TSLP and fibrocytes in the generation of IL-13-induced skin fibrosis. In AD lesion, cessation of IL-13 transgene expression resulted in reduced skin inflammation but with no effect on further progression of fibrosis. This was accompanied by markedly increased CD34(+)/procollagen 1(+) fibrocytes. Furthermore, fibrocytes express TSLP receptor (TSLPR), and TSLP directly promotes PBMC-derived fibrocytes to produce collagen. Neutralization of TSLP or genetic deletion of TSLPR in IL-13 transgenic mice resulted in a significant reduction in fibrocytes and in skin fibrosis. Furthermore, reduction of fibrosis by depletion of TSLP was independent of IL-13. Interestingly, the number of fibrocytes was highly increased in the skin samples of AD patients. These data indicate that the progression of skin fibrosis in IL-13-induced AD occurs via TSLP/TSLPR-dependent but IL-13-independent novel mechanisms by promoting fibrocyte functions.     

MMP9-PEX的原核表达、纯化与复性研究

目的 对人基质蛋白酶9（matrix metalloproteinase,MMP9）C端血红素结合蛋白样结构域（hemopexin domain,PEX）的原核表达条件、透析复性条件进行优化,以获得高表达的活性蛋白.方法 将构建好的原核表达载体PET-his-MMP9-PEX转化至大肠埃希菌菌株BL21（DE-3）,异丙基β-D硫代半乳糖苷（IPTG）诱导后产生包涵体蛋白,探讨不同IPTG浓度、不同诱导时间、不同菌液密度值对目的 蛋白表达产量的影响;盐酸胍裂解包涵体,采用NI-NTA进行纯化,对纯化蛋白的透析复性条件进行优化;采用明胶酶谱的方法检测MMP9-PEX的活性.结果 经纯化透析复性后蛋白的纯度在95%以上,复性回收率为36.86%.结论 本研究优化了MMP9-PEX的原核表达、透析复性条件,获得了有活性的MMP9-PEX蛋白,为进一步研究MMP9-PEX的功能提供了实验基础.     

Role of licochalcone A on thymic stromal lymphopoietin expression: Implications for asthma

Asthma is a common chronic inflammatory disease characterized by the infiltration and accumulation of memory-like Th2 cells and eosinophils. Viral infection has emerged as the most common cause of severe episodes of asthma. For the treatment of bronchial asthma, the root of liquorice (Glycyrrhiza glabra) has been used as a traditional medicine in the East and West. Licochalcone A is the predominant, characteristic chalcone in liquorice root. To determine whether licochalcone A possesses an anti-inflammatory effect, we tested its effect on the expression and production of thymic stromal lymphopoietin (TSLP) in BEAS 2B cells and primary bronchial epithelial cells. We found that polyinosinic-polycytidylic acid (poly-IC)-induced TSLP expression was suppressed by treatment with licochalcone A in a dose- and time-dependent manner. We also found that poly-IC-induced mRNA expression of other proinflammatory mediators such as MCP-1, RANTES, and IL-8 was suppressed by licochalcone A. Furthermore, licochalcone A suppressed poly-IC-induced nuclear factor kappa B (NF-κB) nuclear translocation and DNA-binding activity by suppressing the Iκβ kinase (IKK) activity but not by direct phosphorylation of p65 at serine 276. Collectively, our findings suggest that licochalcone A suppresses poly-IC-induced TSLP expression and production by inhibiting the IKK/NF-κB signaling pathway, which might be involved in the pathogenesis of virus-exacerbated asthma. Further elucidation of the mechanisms underlying these observations can help develop therapeutic strategies for virally induced asthma.     

布地奈德对A549细胞胸腺基质淋巴细胞生成素表达作用的研究

目的:观察细胞因子刺激气道上皮细胞胸腺基质淋巴细胞生成素(TSLP)表达是否涉及核因子κB(NF-κB),并探讨糖皮质激素布地奈德对气道上皮细胞TSLP表达和NF-κB核转位的影响.方法:A549细胞与细胞因子白介素1β(IL-1β)、白介素4(IL-4)和布地奈德共同孵育,以不加任何细胞因子或布地奈德培养的A549细胞为对照组,采用RT-PCR方法测定TSLP mRNA表达,细胞免疫荧光方法检测TSLP和NF-κB的表达情况.结果:与对照组比较,IL-1β(10 ng/ml)及IL-4(10 ng/ml)显著刺激A549细胞TSLP mRNA表达,且NF-κB(p65)核转住增加(均P＜0.05).布地奈德干预后TSLP mRNA的表达和NF-κB(p65)的核转位显著减少(P＜0.05).结论:细胞因子促进气道上皮细胞诱导性表达TSLP与NF-κB激活有关,抑制TSLP表达和NF-κB激活可能是布地奈德治疗哮喘的重要机制.     

Interleukin-32 induced thymic stromal lymphopoietin plays a critical role in the inflammatory response in human corneal epithelium

Interleukin (IL)-32, a novel cytokine, participates in a variety of inflammatory disorders. Thymic stromal lymphopoietin (TSLP) plays important roles in mucosal epithelial cells, especially in allergy-induced inflammation, through the TSLP-TSLPR (thymic stromal lymphopoietin receptor) signalling pathway. However, the association of IL-32 with TSLP on the ocular surface remains unclear. The present work aimed to assess the functional association of IL-32 with TSLP in the control of pro-inflammatory cytokine levels in the corneal epithelium. Human corneal tissue specimens and human corneal epithelial cells (HCECs) were administered different concentrations of IL-32 in the presence or absence of various inhibitors to assess TSLP levels and localization, as well as the molecular pathways that control pro-inflammatory cytokine production. TSLP mRNA levels were determined by real time RT- PCR, while protein levels were quantitated by ELISA and immunohistochemical staining. TSLP protein expression was examined in donor corneal epithelium samples. IL-32 significantly upregulated TSLP and pro-inflammatory cytokines (TNFα and IL-6) in HCECs at the gene and protein levels. The production of pro-inflammatory molecules by IL-32 was increased by recombinant TSLP. Interestingly, both NF-κB (quinazoline) and caspase-1 (VX-765) inhibitors suppressed the IL-32-related upregulation of pro-inflammatory cytokines (TNFα and IL-6). These findings demonstrate that IL-32 and IL-32-induced-TSLP are critical cytokines that participate in inflammatory responses through the caspase-1 and NF-κB signalling pathways in the corneal epithelium, suggesting new molecular targets for inflammatory diseases of the ocular surface. The effects of IL-32 on cell proliferation and apoptosis were investigated by MTT assays and RT-PCR,respectively. The results demonstrated that IL-32 inhibits cells apoptosis in HCECs.     

绿色荧光蛋白的原核表达、纯化以及抗体制备

用PCR扩增出绿色荧光蛋白(GFP)基因,插入到pGEX-KG表达载体中,并将构建出的重组质粒命名为pKG- GFP。将重组载体导入大肠杆菌DH10β中,经IPTG诱导产生GST-GFP融合蛋白,同时以可溶蛋白和包涵体两种形式存在。 GST-GFP分子量大约为53kDa,与其理论值大小一致,用亲和层析以及凝血酶处理纯化GFP。纯化的产物经证实具有很好的 均一性。以GFP免疫新西兰家兔,制备多克隆抗体,Westernblotting测定抗血清效价。