共查询到18条相似文献，搜索用时 984 毫秒
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人白介素-18(human interleukin 18,hIL18)基因与表皮生长因子受体干扰序列(EGFloop C sequence,EGF)构建IL18-EGF融合基因,利pET32a/E.coli BL21(DE3)表达IL18-EGF融合基因,通过纯化获得有活性的靶向的IL18-EGF融合蛋白,并进行体外细胞试验评价IL18-EGF融合蛋白的抗肿瘤活性.结果表明IL18-EGF融合蛋白能促进人PBMNC的增殖,诱导KG-1细胞分泌IFN-γ、促进肿瘤抗原诱导的B细胞、NK及其CD4+T细胞的活化. 相似文献
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目的:设计获得5段hIL-18异构体,定向插入pEGFP-C1质粒形成融合基因,转染胰腺癌Bx-PC-3细胞并表达,为进一步研究改建的hIL-18蛋白功能奠定实验基础.方法:设计引物从pGEM-T-hIL-18质粒中PCR扩增得到5段不同的hIL-18片段,分别连接入pEGFP-C1载体构建不同的突变子(Mu0、Mu1、Mu2、Mu3和Mu4),转染原位胰腺癌BxPC-3细胞,以荧光显微镜观察绿色荧光蛋白的表达,RT-PCR法检测转染细胞中hIL-18表达水平.结果:(1)五种重组质粒经酶切与测序证实构建成功;(2)BxPC-3细胞转染重组质粒后可观察到绿色荧光蛋白的表达.结论:(1)成功构建了hIL-18五种异构体的绿色荧光蛋白表达载体;(2)改建的hIL-18蛋白可与绿色荧光蛋白在BxPC-3细胞融合表达. 相似文献
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《科学中国人》2015,(26)
目的:构建Visfatin原核表达质粒,诱导表达并纯化重组人内脂素(Visfatin)。方法:从人LO2细胞中提取总RNA后,经RT-PCR法得到人Visfatin的c DNA序列,用以构建带HIS标签的Visfatin重组质粒。然后将重组质粒转化到大肠杆菌BL21中,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,用Ni柱亲和层析纯化融合蛋白。结果:PCR扩增出的目的基因片段为1600 bp左右,与理论大小一致。SDS-PAGE、Western blot、质谱分析数据证明了蛋白是Visfatin融合蛋白。结论:成功的表达并纯化得到带HIS标签的Visfatin融合蛋白。 相似文献
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《科学中国人》2015,(26)
目的构建SLO原核表达质粒,重组蛋白的诱导表达并纯化。方法提取链霉菌溶血素O模板DNA,PCR法扩增slo基因。构建融合表达重组质粒p GEX-6p-1-slo和pet32a-tev-slo,将正确的重组质粒转化至大肠杆菌BL21和大肠杆菌BL21-DE3,使用异丙基硫代βD半乳糖苷(IPTG)诱导表达重组融合蛋白。采用亲和层析纯化重组蛋白,切除标签后,再通过亲和层析纯化,获取SLO重组蛋白。结果 PCR扩增出slo基因,基因片段(1700 bp)与理论一致。经SDSPAGE,蛋白质印迹显示重组蛋白相对分子质量为55 k Da,与数据库中的相对分子质量结果相符。结论成功构建了slo原核表达质粒,表达并纯化了SLO重组蛋白。 相似文献
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目的:扩增弓形虫SAG2基因,构建pGEX-4T-1-SAG2重组质粒,在大肠杆菌中表达。方法:设计并合成引物,经PCR法扩增SAG2基因序列,与pMD18-T载体连接,构建pMD18-T-SAG2质粒,经酶切鉴定、测序,与pGEX-4T-1载体连接,构建pGEX-4T-1-SAG2质粒。将重组质粒转入大肠杆菌诱导表达,进行SDS-PAGE和Western-blot鉴定。结果:扩增出约561bp的SAG2基因序列,成功构建pGEX-4T-1-SAG2质粒,并在大肠杆菌中得到高效表达,融合蛋白的表达量占菌体总蛋白的40%。SDS-PAGE电泳显示pGEX-4T-1-SAG2的融合蛋白的分子量约为45kd。Western-blot显示融合蛋白有良好的抗原性。结论表达质粒在大肠杆菌中得到高效表达,为进一步研究弓形虫病的诊断做好准备。 相似文献
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目的探讨小鼠肝癌树突状细胞融合瘤苗抗肿瘤作用及其机制。方法用PEG法制备小鼠肝癌树突状细胞融合瘤苗;流式细胞仪检测融合细胞表型特征;RT-PCR法检测肿瘤组织中TNF-αmRNA、IFN-γmRNA表达:Western blot法检测肿瘤组织中Bcl-2、Bax、Caspase-3蛋白表达。结果小鼠肝癌树突状细胞融合瘤苗具备树突状细胞及肝癌细胞表型特征,能显著促进肿瘤组织中TNF-αmRNA、IFN-γm-RNA及Bcl-2、Bax、Caspase-3蛋白表达。结论小鼠肝癌树突状细胞融合瘤苗能有效地诱导抗肿瘤免疫反应,促进肿瘤细胞凋亡,在预防和治疗肝癌的复发及转移过程中有广阔的应用前景。 相似文献
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根据鸡白细胞介素18（IL-18）cDNA基因序列设计了1对特异性引物，应用RT-PCR技术，从脂多糖（lipopolysaccharide，LPS）刺激的我国地方品种萧山鸡原代鸡脾细胞中扩增并克隆鸡IL-18全长基因（Genbank accession，AY628648）。扩增片段全长591bp，共编码197个氨基酸的前体蛋白，其中含有表达完整功能蛋白所必需的起始密码子和终止密码子。该序列与国外报道的鸡IL-18全长基因核苷酸序列及推导的氨基酸序列的同源性为98．99％。只在引导序列中出现两个氨基酸残基的缺失，分子进化分析表明萧山鸡IL-18基因与火鸡以及家鸭基因形成一个独立的分支。将萧山鸡IL-18成熟蛋白序列插入pGEX-4T-2载体并在Ecoli中得到表达，获得45kD的GST-IL-18融合蛋白。经纯化后用于制备多克隆抗体。本研究对萧山鸡白细胞介素18的全长基因进行了克隆及表达，并对其分子进化进行了分析。为深入研究其功能奠定了基础。 相似文献
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Renu Yadav Babban Jee Sudhir Kumar Awasthi 《Indian journal of clinical biochemistry : IJCB》2015,30(1):109-112
Curcumin is a major bioactive compound of turmeric that exerts its anti-inflammatory effects by suppressing the many pro-inflammatory cytokines and chemokines in a number of cell types and pathologic conditions. Interleukin-18 (IL-18) is a novel pro-inflammatory cytokine which plays an important role not only in generating Th1 responses but also in inducing severe inflammatory reactions. As curcumin induced inhibition of IL-18 production in keratinocytes and mice is well known, effect of curcumin on IL-18 release in macrophages remains unknown. Hence, this present study has been designed to evaluate the effect of curcumin on IL-18 production and necrotic cell death in murine macrophages-like cells treated with or without lipopolysaccharide (LPS). The IL-18 secretion in cell culture supernatants was assayed by enzyme-linked immunosorbent assay and cytotoxicity was determined by lactate dehydrogenase release assay. Our results demonstrate that curcumin significantly inhibited the production of pro-inflammatory cytokine IL-18 in E.coli LPS stimulated murine macrophage-like cells RAW264.7 in a concentration-dependent manner. Interestingly, curcumin had no cytotoxic effect on murine macrophage-like cells. Our findings suggest that curcumin may be used as a potential therapeutic agent for the treatment of inflammatory diseases. 相似文献
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SRB显色法用于抗癌药物敏感性试验的研究 总被引：13，自引：0，他引：13
采用ＳＲＢ显色法对９６孔培养板中短期培养的人大肠癌细胞株Ｃｏｌｏ２０５、人骨肉瘤细胞株ＯＳ７３２,人舌癌细胞株Ｔｃａ８１１３经５－ＦＵ,顺铂,蟾酥等药物处理后的活性进行了研究。结果表明,ＳＲＢ法对这些细胞株的体外抗癌药物敏感试验结果稳定,该法与其它细胞毒试验相比有一定优点,值得推广应用。 相似文献
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BackgroundChinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE.ResultsHigher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa).ConclusionsThese results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.How to cite: Pérez-Rodriguez S, Ramírez OT, Trujillo-Roldán MA et al. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.006. 相似文献
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Definable surface chemistry is essential for many applications of microfluidic polymer systems. However, small cross-section channels with a high surface to volume ratio enhance passive adsorption of molecules that depletes active molecules in solution and contaminates the channel surface. Here, we present a one-step photochemical process to coat the inner surfaces of closed microfluidic channels with a nanometer thick layer of poly(ethylene glycol) (PEG), well known to strongly reduce non-specific adsorption, using only commercially available reagents in an aqueous environment. The coating consists of PEG diacrylate (PEGDA) covalently grafted to polymer surfaces via UV light activation of the water soluble photoinitiator benzoyl benzylamine, a benzophenone derivative. The PEGDA coating was shown to efficiently limit the adsorption of antibodies and other proteins to <5% of the adsorbed amount on uncoated polymer surfaces. The coating could also efficiently suppress the adhesion of mammalian cells as demonstrated using the HT-29 cancer cell line. In a subsequent equivalent process step, protein in aqueous solution could be anchored onto the PEGDA coating in spatially defined patterns with a resolution of <15 μm using an inverted microscope as a projection lithography system. Surface patterns of the cell binding protein fibronectin were photochemically defined inside a closed microfluidic device that was initially homogeneously coated by PEGDA. The resulting fibronectin patterns were shown to greatly improve cell adhesion compared to unexposed areas. This method opens for easy surface modification of closed microfluidic systems through combining a low protein binding PEG-based coating with spatially defined protein patterns of interest. 相似文献
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BackgroundChinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells.ResultsIn this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95–100% of productivity after 40 days of continuous culture (~ 48–56 generations).ConclusionsSuspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.How to cite: Li J, Wei S, Cao C, et al. Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines. Electron J Biotechnol 2019;41. https://doi.org/10.1016/j.ejbt.2019.07.002. 相似文献
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Namdev S. Togre Priyanka S. Bhoj Vishal K. Khatri Aditya Tarnekar Kalyan Goswami Moreshwar R. Shende M. V. R. Reddy 《Indian journal of clinical biochemistry : IJCB》2018,33(3):282-289
Helminthic infections lead to the release of various molecules which play an important role in modulation of the host immune system. Such filarial proteins with immunomodulatory potential can be used for therapeutic purpose in inflammatory and immune mediated diseases. In the present study, we have explored the prophylactic effect of filarial SXP–RAL family protein of Wuchereria bancrofti i.e. rWbL2 protein in DSS induced inflammatory ulcerative colitis in a mouse model. Prior treatment of rWbL2, followed by induction of colitis, showed significantly reduced disease severity as indicated by the decreased disease manifestations and improved macroscopic and microscopic inflammation. This preventive effect was found to be associated with increased release of anti-inflammatory cytokine IL-10 and decreased release of proinflammatory cytokines IFN-γ, TNF-α, IL-6 and IL-17 by the splenocytes of treated mice. From this study, it can be envisaged that pretreatment with filarial protein, rWbL2, can prevent the establishment of ulcerative colitis in BALB/c mice. The underlying immunological mechanism may involve the up-regulation of Th2 immune response with down-regulation of Th1 response. 相似文献