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TIM-1-Fc融合蛋白对哮喘小鼠Th1/Th2和Th17/Treg免疫失衡的调节
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作者 曹津萌 卿吉琳 +4 位作者 朱莉雅 魏燕 赵艺莲 叶超 陈治中 《华中科技大学学报(医学版)》 CAS CSCD 北大核心 2024年第4期479-486,527,共9页
目的制备T细胞免疫球蛋白和黏蛋白结构域1(T cell immunoglobulin domain and mucin domain protein-1,TIM-1)-Fc融合蛋白并探讨TIM-1-Fc融合蛋白对卵白蛋白(ovabumin,OVA)诱导的哮喘小鼠的干预作用及潜在作用机制。方法通过基因工程技... 目的制备T细胞免疫球蛋白和黏蛋白结构域1(T cell immunoglobulin domain and mucin domain protein-1,TIM-1)-Fc融合蛋白并探讨TIM-1-Fc融合蛋白对卵白蛋白(ovabumin,OVA)诱导的哮喘小鼠的干预作用及潜在作用机制。方法通过基因工程技术获得TIM-1-Fc融合蛋白。采用腹腔注射OVA氢氧化铝溶液致敏建立过敏性哮喘小鼠模型,随机分为对照组、哮喘组和TIM-1-Fc干预组。每次干预前20 min,使用40μL卵白蛋白生理盐水溶液(OVA-NS)滴鼻,TIM-1-Fc融合蛋白干预包括TIM-1-Fc滴鼻组(每只小鼠每次给予1μg/40μL TIM-1-Fc滴鼻)和TIM-1-Fc注射组(每只小鼠每次给予6μg/200μL TIM-1-Fc腹腔注射),每天1次,连续7 d,对照组用生理盐水替代。采用苏木精-伊红(HE)染色观察肺组织病理变化;采用流式细胞术检测小鼠外周血中辅助性T细胞2(type 2 T helper cells,Th2)、辅助性T细胞17(type 17 T helper cells,Th17)和调节性T细胞(Treg)比例及相关细胞因子水平。结果成功构建TIM-1-Fc融合蛋白,成功构建OVA诱导的过敏性哮喘小鼠模型。与哮喘组相比,TIM-1-Fc融合蛋白干预后显著减轻了哮喘小鼠气道炎性损伤和肺组织损伤;TIM-1-Fc融合蛋白干预能显著降低外周血中TIM-1^(+)CD4^(+)T细胞和TIM-1^(+)Th17细胞比例,使TIM-1^(+)Treg细胞增多,显著降低Th2、Th17细胞比例,提高Treg细胞比例,调节哮喘中Th1/Th2和Th17/Treg免疫失衡。结论TIM-1-Fc融合蛋白改善OVA诱导的过敏性哮喘小鼠气道炎症和肺组织损伤,其作用机制可能与TIM-1-Fc融合蛋白对Th1/Th2和Th17/Treg的免疫调节有关。 展开更多
关键词 TIM-1 TIM-1-Fc融合蛋白 过敏性哮喘 TH1/TH2 TH17/TREG
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GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1 被引量:1
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作者 LI Jiang YANG Nan-yang +5 位作者 GUAN Xin-gang ZHANG Shu-zhi ZHANG Yan QIN Mei-ling MA Tong-hui LI Xiao-meng 《Chemical Research in Chinese Universities》 SCIE CAS CSCD 2009年第4期500-505,共6页
Aquaporins(AQPs) are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C... Aquaporins(AQPs) are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1(AQP1) as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1(DNA sequence from 700 to 801 bp) recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein. 展开更多
关键词 Aquaporin 1 GST fusion protein Polyclonal antibody Gene knockout mice Membrane protein
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蛋白激酶全抑制分析揭示KG-1细胞增殖的分子机制
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作者 段毓 徐凝馨 +6 位作者 曹琼 杨恺 王金娟 刘思瑾 贾峰峰 刘建兵 李莉 《中国临床药理学与治疗学》 CAS CSCD 北大核心 2024年第6期621-628,共8页
目的:通过分析KG-1细胞对各种蛋白激酶抑制剂的反应,探讨其增殖的分子机制。方法:采用CCK-8法、实时荧光定量PCR(qRT-PCR)和Western-blot检测各种蛋白激酶抑制剂对KG-1细胞增殖、相关基因mRNA表达水平以及FGFR1下游信号通路蛋白磷酸化... 目的:通过分析KG-1细胞对各种蛋白激酶抑制剂的反应,探讨其增殖的分子机制。方法:采用CCK-8法、实时荧光定量PCR(qRT-PCR)和Western-blot检测各种蛋白激酶抑制剂对KG-1细胞增殖、相关基因mRNA表达水平以及FGFR1下游信号通路蛋白磷酸化水平的影响。结果:NVP-BGJ398和PD173074有效抑制KG-1细胞的增殖,表明FGFR及其下游信号通路在KG-1细胞增殖过程中具有关键作用。使用FGFR抑制剂处理后,p-FGFR1和p-STAT5水平显著下降(P<0.001),p-Akt水平稍有下降(P<0.05),并未影响p-ERK水平(P>0.05)。结论:FGFR1OP2-FGFR1主要作用于下游STAT5信号通路,以促进细胞增殖。蛋白激酶全抑制分析是一种可靠而直接的方法,可用于确定癌细胞增殖的分子机制。 展开更多
关键词 KG-1细胞 蛋白激酶抑制剂 FGFR1OP2-FGFR1融合基因 STAT5
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子宫内膜样腺癌组织NuSAP1、MFN2表达与临床病理特征和预后的关系
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作者 谢旭 张欣萍 +1 位作者 郭丽萍 许欣 《海南医学》 CAS 2024年第12期1709-1713,共5页
目的探讨子宫内膜样腺癌(EA)组织核仁纺锤体相关蛋白1(NuSAP1)、线粒体融合蛋白2(MFN2)表达与临床病理特征和预后的关系。方法选取2019年3月至2020年10月南阳市第一人民医院收治的121例EA患者作为研究对象,应用免疫组织化学染色法检测... 目的探讨子宫内膜样腺癌(EA)组织核仁纺锤体相关蛋白1(NuSAP1)、线粒体融合蛋白2(MFN2)表达与临床病理特征和预后的关系。方法选取2019年3月至2020年10月南阳市第一人民医院收治的121例EA患者作为研究对象,应用免疫组织化学染色法检测患者癌组织及其对应癌旁组织NuSAP1、MFN2阳性表达率,分析癌组织NuSAP1、MFN2阳性表达率与患者临床病理特征的关系。出院后随访3年,完成随访112例,应用Kaplan-Meier生存曲线分析NuSAP1、MFN2阳性表达组和阴性表达组的预后差异,应用多因素COX回归分析EA患者预后的影响因素。结果EA癌组织NuSAP1阳性表达率为72.37%,明显高于癌旁组织的35.54%,MFN2阳性表达率为38.02%,明显低于癌旁组织的80.17%,差异均有统计学意义(P<0.05);国际妇产科联盟(FIGO)临床分期Ⅱ~Ⅲ期、中低分化、有淋巴结转移、深肌层浸润癌组织中NuSAP1阳性表达率分别为88.68%、83.54%、96.15%、90.63%,明显高于FIGO临床分期Ⅰ期的60.29%、高分化的57.14%、无淋巴结转移的55.79%、浅肌层浸润癌组织的66.29%,差异均有统计学意义(P<0.05);FIGO临床分期Ⅱ~Ⅲ期、中低分化、有淋巴结转移、深肌层浸润癌组织中MFN2阳性表达率分别为18.87%、30.38%、7.69%、15.63%,明显低于FIGO临床分期Ⅰ期的52.94%、高分化的52.38%、无淋巴结转移的46.32%、浅肌层浸润癌组织的46.07%,差异均有统计学意义(P<0.05);Kaplan-Meier法分析结果显示,NuSAP1阴性表达组和MFN2阳性表达组患者的3年生存率分别为90.00%和87.80%,明显高于NuSAP1阳性表达组的69.51%和MFN2阴性表达组的67.61%,差异均有统计学意义(P<0.05)。结论EA患者组织中NuSAP1阳性表达率升高,MFN2阳性表达率降低,NuSAP1、MFN2表达水平与患者肿瘤分化、FIGO临床分期、淋巴结转移及深肌层浸润有关,且是患者死亡的危险因素,提示NuSAP1、MFN2可能参与了EA患者的疾病进展。 展开更多
关键词 子宫内膜样腺癌 核仁纺锤体相关蛋白1 线粒体融合蛋白2 临床病理特征 预后
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Construction,Expression and Purification of SUMO1-GST Fusion Protein
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作者 QIAO Xiao-fang FANG Xue-dong LIU Jun 《Chemical Research in Chinese Universities》 SCIE CAS CSCD 2011年第2期245-248,共4页
Sumoylation is an important protein modification discovered recently. SUMO(small ubiquitin-related modifier) pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein... Sumoylation is an important protein modification discovered recently. SUMO(small ubiquitin-related modifier) pathway regulates the protein stability and transcriptional activity with a 12-kDa small molecular protein, SUMO, ligated to the target protein. The purification of SUMO proteins is a key step to reveal their function. The purpose of this study was to construct the recombinant SUMO1 gene cloned to a pGEX-4T-1 vector to express and purify the SUMO1-GST fusion protein in Escherichia coli. First, the full length DNA sequence of SUMO1 gene was amplified by PCR and was ligated to pMD18-T vector. Then the SUMO1 gene was subcloned to pGEX-4T-1 prokaryotic expression vector between BamHI and XhoI sites, and transformed in Escherichia coli DH5α cells. The right colonies were identified by restrictive enzyme digestion and sequencing. The correct rebombinant plasmid of pGEX-4T-1-SUMO1 was transformed in Escherichia coli BL21 cells and then induced by IPTG(isopropyl- β-D-1- thiogalacto-pyranoside) to express the SUMO1-GST fusion protein. The highly purified SUMO1-GST(glutathione S-transferase) fusion protein was obtained by affinity chromatography. Finally, the properties of SUMO1-GST fusion protein were confirmed by Coomassie brilliant blue strain and Western blot analysis. The recombinant plasmid of pGEX-4T-1-SUMO1 was successfully constructed, and SUMO1-GST fusion proteins were successfully expressed. 展开更多
关键词 Small ubiquitin-related modifier 1 (SUMO1 Gutathione S-transferase(GST) fusion protein Affinity chromatography
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Expression and characterization of Mac-1-FP fusion protein in CHO cells
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作者 刁飞 严鸣 +3 位作者 朱晓燕 杨勇骥 刘辉 徐仁宝 《Journal of Medical Colleges of PLA(China)》 CAS 2004年第6期321-324,共4页
Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalia... Objective: To construct mammalian cell expression vectors for Mac-1 with CFP and YFP and apply FRET to study the dimerization and function of CD11 b( Mac-1 α subunit) and CD18(Mac-1 β subunit). Methods: The mammalian cell expression vector for CD11b fused with CFP at the carboxyl terminal was constructed to create recombinant plasmid of pCD11b-CFP. Then pCD11b-CFP was co-transfected with pYFP-CD18 into CHO cell, a fibroblast like cell line, as a target cell within which there are some signal pathways involved in inflammatory stimulation but without endogenous Mac-1. Then CHO cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were selected by Western blot and laser scanning confocal microscope. Results: The cyan and yellow fluorescence in co-transfected positive CHO cells were observed under a fluorescence microscope. CHO-Mac-1-FP cells stably expressing both CD11b-CFP and YFP-CD18 fusion proteins were obtained as demonstrated by Western blot successfully. The adhesive activity of CHO-Mac-1-FP cells with CHO-1CAM-1 cells was increased markedly by treatment with PMA, suggesting the translocation of GD11b-CFP and YFP-CD18 to the plasma membrane in CHO-Mac-1-FP cells and dimerization of CD11b-CFP and YFP-CD18 just as the function of the wild type Mac-1. Conclusion: CHO-Mac-1-FP cells with adhesive activity are established successfully, thus CHO-Mac-1-FP cells may be useful for the study of Mac-1 by FRET and for other purposes. 展开更多
关键词 MAC-1 cyan fluorescent protein yellow fluorescent protein fusion protein
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XCL1/Glypican-3 fusion protein induces potent CD8^+ T-cell generation and enhances the anti-PD1 effect to suspend hepatocellular carcinoma development
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作者 Kun Chen Yanmei Wang +6 位作者 Hong Zhao Zhiyuan Wu Xinyu Bi Jianguo Zhou Dongmei Wang Jianqiang Cai Chunfeng Qu 《Cancer Biology & Medicine》 SCIE CAS CSCD 2018年第S01期17-18,共2页
Objective:To induce potent CD8^+T-cells against glypican-3(GPC3),which is overexpressed in hepatocellular carcinoma(HCC),to suspend tumor development.Methods:Since the chemokine receptor XCR1 is selectively expressed ... Objective:To induce potent CD8^+T-cells against glypican-3(GPC3),which is overexpressed in hepatocellular carcinoma(HCC),to suspend tumor development.Methods:Since the chemokine receptor XCR1 is selectively expressed on professional cross-presenting CD8α^+dendritic cells(DCs). 展开更多
关键词 XCL1/Glypican-3 fusion protein
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Roles of low?density lipoproteinreceptor?related protein 1 in tumors 被引量:5
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作者 Peipei Xing Zhichao Liao +5 位作者 Zhiwu Ren Jun Zhao Fengju Song Guowen Wang Kexin Chen Jilong Yang 《Chinese Journal of Cancer》 SCIE CAS CSCD 2016年第1期4-11,共8页
Low-density lipoprotein receptor-related protein 1(LRP1,also known as CD91),a multifunctional endocytic and cell signaling receptor,is widely expressed on the surface of multiple cell types such as hepatocytes,fibrobl... Low-density lipoprotein receptor-related protein 1(LRP1,also known as CD91),a multifunctional endocytic and cell signaling receptor,is widely expressed on the surface of multiple cell types such as hepatocytes,fibroblasts,neurons,astrocytes,macrophages,smooth muscle cells,and malignant cells.Emerging in vitro and in vivo evidence demonstrates that LRP1 is critically involved in many processes that drive tumorigenesis and tumor progression.For example,LRP1 not only promotes tumor cell migration and invasion by regulating matrix metalloproteinase(MMP)-2and MMP-9 expression and functions but also inhibits cell apoptosis by regulating the insulin receptor,the serine/threonine protein kinase signaling pathway,and the expression of Caspase-3.LRPI-mediated phosphorylation of the extracellular signal-regulated kinase pathway and c-jun N-terminal kinase are also involved in tumor cell proliferation and invasion.In addition,LRP1 has been shown to be down-regulated by microRNA-205 and methylation of LRP1CpG islands.Furthermore,a novel fusion gene,LRP1-SNRNP25,promotes osteosarcoma cell invasion and migration.Only by understanding the mechanisms of these effects can we develop novel diagnostic and therapeutic strategies for cancers mediated by LRP1. 展开更多
关键词 LOW-DENSITY LIPOprotein receptor-related protein 1 Tumorigenesis Invasion migration Proliferation apoptosis Signaling pathway MicroRNA fusion gene
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Stability improvement of human collagenα1(I)chain using insulin as a fusion partner 被引量:2
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作者 Yu Mi Yuan Gao +5 位作者 Daidi Fan Zhiguang Duan Rongzhan Fu Lihua Liang Wenjiao Xue Shanshan Wang 《Chinese Journal of Chemical Engineering》 SCIE EI CAS CSCD 2018年第12期2607-2614,共8页
To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted int... To enhance the stability of recombinant human collagen α1(I) chains(rhCOL1 A1) in production and purification stages, a gene fragment fusing COL1 A1 and insulin protein coding domains was synthesized and inserted into the pPIC9 K expression vector. The fusion peptide-expressing Pichia pastoris strain was created by transformation.After optimization of shake flask cultures, the ultimate intracellular expression level of the insulin-collagen α1(I) chain fusion protein(INS-COL1 A1) reached about 300 mg·L^(-1), and no obvious protein degradation was found in the fermentation and purification processes. The His-tagged recombinant fusion protein was detected by western blotting and was effectively purified using Ni^(2+)-chelating chromatography. A prominent improvement in the stability of INS-COL1 A1 was observed compared to rhCOL1 A1 in vitro, and the rhCOL1 A1 released from the fusion protein was studied by LC–MS/MS and in bioassays. The results showed that the purified rhCOL1 A1 was consistent with the native protein in amino acid composition and had a similar biological compatibility. To our knowledge, this is the first study to demonstrate the use of insulin as a fusion protein to improve the stability of easily degradable proteins. 展开更多
关键词 fusion expression HUMAN COLLAGEN α1(I) CHAIN INSULIN protein STABILITY Pichia pastoris
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滋肾活血方对血管性痴呆大鼠分裂与融合蛋白Mfn1、Mfn2、Drp1、Fis1的影响 被引量:2
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作者 秦茂 伍大华 +1 位作者 张秀丽 谢乐 《湖南中医药大学学报》 CAS 2023年第1期21-26,共6页
目的观察滋肾活血方对血管性痴呆(vascular dementia,VD)大鼠海马组织线粒体融合蛋白1(mitofusin 1,Mfn1)、线粒体融合蛋白2(mitofusin 2,Mfn2)、线粒体动力蛋白相关蛋白1(dynamin-related protein 1,Drp1)、线粒体分裂蛋白1(fission mi... 目的观察滋肾活血方对血管性痴呆(vascular dementia,VD)大鼠海马组织线粒体融合蛋白1(mitofusin 1,Mfn1)、线粒体融合蛋白2(mitofusin 2,Mfn2)、线粒体动力蛋白相关蛋白1(dynamin-related protein 1,Drp1)、线粒体分裂蛋白1(fission mitochondrial 1,Fis1)表达的影响。方法选用雄性SD大鼠60只,随机均分为假手术组、模型组、滋肾活血高剂量组、滋肾活血中剂量组、滋肾活血低剂量组、西药组。除假手术组外,其余各组均采用改良2-VO法建立VD大鼠模型。每组大鼠按9 mL/(kg·d)剂量灌胃相应药物。模型组、假手术组给予蒸馏水,滋肾活血低剂量组、滋肾活血中剂量组、滋肾活血高剂量组予以滋肾活血方溶液[9.8、17.8、35.6 g/(kg·d)]灌胃,西药组以多奈哌齐溶液[150 mg/(kg·d)]灌胃。连续喂药2周后,采用水迷宫实验评估大鼠学习记忆功能;取海马组织,采用免疫组化法检测实验大鼠海马组织中的Mfn1、Mfn2、Drp1、Fis1蛋白表达量。结果与假手术组比较,模型组大鼠逃避潜伏期(escape latency,EL)延长(P<0.05),Mfn1、Mfn2、Drp1蛋白的表达量降低(P<0.05)。与模型组对比,滋肾活血中、高剂量组大鼠EL缩短(P<0.05),Mfn1、Mfn2、Drp1蛋白表达量升高(P<0.05)。与滋肾活血低剂量组比较,滋肾活血中、高剂量组大鼠EL缩短(P<0.05),Mfn1、Drp1蛋白表达量升高(P<0.05)。结论滋肾活血方可能通过调节细胞内线粒体分裂与融合,上调Mfn1、Mfn2、Drp1蛋白的表达,从而改善认知功能。 展开更多
关键词 血管性痴呆 滋肾活血方 线粒体分裂 线粒体融合 线粒体融合蛋白1 线粒体融合蛋白2 线粒体动力蛋白相关蛋白1 线粒体分裂蛋白1
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Expression of GST-IL-1 fusion gene
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作者 陈梅红 王字玲 +3 位作者 邓健蓓 赵忠良 陈南春 苏成芝 《Journal of Medical Colleges of PLA(China)》 CAS 1996年第2期79-83,共5页
Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ... Two GST-IL-1 fusion genes were constructed by inserting different cDNA fragments of human interleukin1 (IL-1) into the 3'-terminus of GST gene in the fusion protein expression vector pGEX-4T. After IPTG induction ,SDS-PAGE was employed to detect the gene expression. No corresponding protein encoded by GST gene fused with the whole-length 816 bp IL-1 cDNA was observed, nor was free GST protein. However, the fusion protein of GST and IL-1 cDNA without the 189 bp at the 5'- terminus was detected, amounting to 30% of the total bacterial protein expressed. This might suggest that the sequence of 1-189 bp of IL-1 cDNA affected the expression of the fusion gene. That is to say, the downstream sequence distant from the translation start codon AUG in the target gene could significantly affect the expression of the fusion gene. 展开更多
关键词 gene EXPRESSION fusion protein INTERLEUKIN-1 GLUTATHIONE-S-TRANSFERASE
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Mechanisms of microRNA-150, cyclin B1 and mitochondrial-associated protein 2 in regulating apoptosis and inhibiting invasion and migration of Huh-7 hepatocellular carcinoma cells
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作者 Feng Wen Yan Xiang 《Journal of Hainan Medical University》 2019年第9期11-14,共4页
Objective: To explore the mechanisms of microRNA-150, cyclin B1 and mitochondrial-associated protein 2 in regulating the apoptosis and inhibiting the invasion and migration of Huh-7 cells. Methods: Huh-7 cells were di... Objective: To explore the mechanisms of microRNA-150, cyclin B1 and mitochondrial-associated protein 2 in regulating the apoptosis and inhibiting the invasion and migration of Huh-7 cells. Methods: Huh-7 cells were divided into the control group, the negative control group (NC group) and the miR-150 overexpression group (mimic group). The miR-150 overexpressing cell line was constructed by plasmid transfection. The cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The cell migration and invasion capacity were measured by cell wound scratch assay and Transwell. The levels of miRNA and mRNA were detected by real-time quantitative polymerase chain reaction and the relative expression levels of proteins were detected by Western blot. Results: MiR-150 significantly inhibited the cell viability of Huh-7 and promoted its apoptosis (P<0.01). After 24 h of cultivation, the mobility of the control group and the NC group were (83.54±4.66)%and (85.57±4.74)%, respectively. The mobility of the mimic group was (49.63±3.78)%, which was significantly lower than that of the control group and the NC group (P<0.01). After 24 h of cultivation, the invasive rate of the control group and the NC group were (100.56±2.87)%and (101.63±3.74)%, respectively, and the invasive rate of mimic group was (51.63±5.32)%, which was significantly lower than that of the control group and the NC group (P<0.01). The expression levels of cyclin B1 protein and mRNA in the mimic group were significantly lower than those in the control group and the NC group (P<0.01), and the level of mitochondrial-associated protein 2 in the mimic group was significantly higher than that in the control group and the NC group (P<0.01). Conclusions: MiR-150 may inhibit the proliferation, migration, invasion and apoptosis of hepatoma carcinoma cell by regulating cyclin B1 or up-regulating mitochondrial-associated protein 2 levels. 展开更多
关键词 Liver cancer MiR-150 CYCLIN B1 MITOCHONDRIAL fusion protein 2
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先天性胫骨假关节的临床治疗新进展
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作者 吴春星 宁波 《外科研究与新技术(中英文)》 2024年第3期181-185,共5页
先天性胫骨假关节是罕见的先天性胫腓骨骨骼畸形,以骨不连、假关节形成、肢体成角短缩等畸形为主要表现,多合并神经纤维瘤病,治疗极其困难。近年来,随着对先天性胫骨假关节发病机制研究的深入,新型手术方法如胫腓骨“4合1融合术”、“Cr... 先天性胫骨假关节是罕见的先天性胫腓骨骨骼畸形,以骨不连、假关节形成、肢体成角短缩等畸形为主要表现,多合并神经纤维瘤病,治疗极其困难。近年来,随着对先天性胫骨假关节发病机制研究的深入,新型手术方法如胫腓骨“4合1融合术”、“Cross-union内固定法”(胫腓骨交叉融合术)的开展,结合新型药物如双膦酸盐、骨形态发生蛋白的综合使用,其骨愈合率逐渐提高,再骨折发生率降低。本文总结了近年来先天性胫骨假关节治疗的现状及进展,以期为临床治疗提供借鉴和思考。 展开更多
关键词 先天性胫骨假关节 4合1融合术 Cross-union内固定法 胫腓骨交叉融合术 骨形态发生蛋白
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胸腺素α_1 基因的克隆表达及其生物活性 被引量:16
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作者 石继红 张英起 +4 位作者 赵永同 赵宁 朱宝娥 颜真 韩苇 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2001年第3期344-349,共6页
胸腺素α1(thymosinalpha 1 ,Tα1)作为一种免疫增强剂 ,临床用途广泛 .为大量制备Tα1,按大肠杆菌惯用密码子合成Tα1基因 ,克隆于质粒pUC1 9的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后 ,串联为 4串体 (Tα1④ ) ,经再次测序确认后克... 胸腺素α1(thymosinalpha 1 ,Tα1)作为一种免疫增强剂 ,临床用途广泛 .为大量制备Tα1,按大肠杆菌惯用密码子合成Tα1基因 ,克隆于质粒pUC1 9的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后 ,串联为 4串体 (Tα1④ ) ,经再次测序确认后克隆入pThioHisA的EcoRⅠ和PstⅠ位点 .转化大肠杆菌T0P1 0 ,酶切鉴定正确后 ,经 1mmol LIPTG诱导 4h ,获得硫氧还蛋白与Tα1④的融合表达 ,用离子交换层析纯化融合蛋白 .溴化氰裂解融合蛋白 ,释放出Tα1单体 ,经离子交换色谱纯化出Tα1.采用3 H TdR参入法进行生物活性测定 ,证实融合蛋白和Tα1均具有刺激小鼠脾淋巴细胞分裂增殖的能力 . 展开更多
关键词 胸腺素Α1 基因克隆 融合表达 蛋白质纯化 生物活性
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重组人胰高血糖素样肽-1的表达、纯化及其生物学活性 被引量:11
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作者 张志珍 杨生生 毛积芳 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2002年第1期5-8,共4页
为获得重组人胰高血糖素样肽 1[recombinanthumanglucagon likepeptide 1(7~ 37) ,rhGLP 1]并研究其生物学活性 ,采用亚磷酸二酯法合成hGLP 1cDNA的 6个寡核苷酸片段 ,拼接成完整的hGLP 1cDNA ,构建重组质粒pGEX hGLP 1,转化大肠杆菌B... 为获得重组人胰高血糖素样肽 1[recombinanthumanglucagon likepeptide 1(7~ 37) ,rhGLP 1]并研究其生物学活性 ,采用亚磷酸二酯法合成hGLP 1cDNA的 6个寡核苷酸片段 ,拼接成完整的hGLP 1cDNA ,构建重组质粒pGEX hGLP 1,转化大肠杆菌BL2 1(DE3)获得表达菌株 .高密度发酵培养的菌体超声破碎后 ,裂解液用Glutathione Sepharose 4B亲和层析纯化得到GST融合蛋白 .经CNBr裂解、QAE SepharoseFF柱层析和脱盐 ,得到纯度大于 90 %的rhGLP 1,质谱测定分子量结果与理论值一致 .生物学活性分析表明 ,rhGLP 1具有明显的降血糖活性 . 展开更多
关键词 重组人胰高血糖素样肽-1 融合蛋白 纯化 生物学活性 表达
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屋尘螨变应原Der p 1基因原核表达产物的纯化及特性鉴定 被引量:9
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作者 刘志刚 杨慧 +3 位作者 付颖媛 高波 朱健琦 吉坤美 《热带医学杂志》 CAS 2006年第6期656-659,共4页
目的建立屋尘螨主要变应原Derp1蛋白原核表达产物的纯化方法,获得理想的表达产物并鉴定其免疫原性。方法大量诱导表达含pET24a-Derp1质粒的BL21工程菌,表达产物以重组蛋白包涵体的形式存在,包涵体经洗涤与溶解后,使用结合6组氨酸的镍柱... 目的建立屋尘螨主要变应原Derp1蛋白原核表达产物的纯化方法,获得理想的表达产物并鉴定其免疫原性。方法大量诱导表达含pET24a-Derp1质粒的BL21工程菌,表达产物以重组蛋白包涵体的形式存在,包涵体经洗涤与溶解后,使用结合6组氨酸的镍柱进行亲和层析纯化蛋白质,用稀释复性方法进行重组蛋白的复性,再用屋尘螨过敏性哮喘患者阳性血清经Dot-ELISA方法分析Derp1纯化蛋白的抗原活性。结果分步洗涤可有效去除重组蛋白包涵体沉淀中混杂的多数杂蛋白成分,亲和层析分离可获得高纯度重组变应原Derp1蛋白。Dot-ELISA法检测结果表明经复性并纯化的Derp1蛋白呈强阳性反应,最佳血清稀释度为1∶64,而重组蛋白与正常人血清呈阴性反应。结论纯化后的融合蛋白Derp1具有较高的纯度及较强的免疫活性,可望作为有效的屋尘螨变应原诊断试剂和疫苗的候选分子。 展开更多
关键词 屋尘螨 变应原 Der P 1 融合蛋白 复性 纯化
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人肌纤生成调节因子1融合蛋白在大肠杆菌的表达和抗体制备 被引量:8
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作者 李天伯 胡洋 +2 位作者 冯爽 左增艳 王以光 《中国医学科学院学报》 CAS CSCD 北大核心 2005年第1期42-47,共6页
目的研究人肌纤生成调节因子1(MR-1)的表达,获得MR-1蛋白,制备MR-1抗体,为MR-1生物功能研究提供基础。方法利用大肠杆菌质粒pGEX-5X-1、pET30a(+)及pET24a(+),分别构建MR-1及其两端与不同标签序列融合的表达载体。在大肠杆菌BL21(DE3)和... 目的研究人肌纤生成调节因子1(MR-1)的表达,获得MR-1蛋白,制备MR-1抗体,为MR-1生物功能研究提供基础。方法利用大肠杆菌质粒pGEX-5X-1、pET30a(+)及pET24a(+),分别构建MR-1及其两端与不同标签序列融合的表达载体。在大肠杆菌BL21(DE3)和BL21-CodonPlus(DE3)-RIL中比较N端和/或C端融合标签序列对该基因表达的影响。通过凝胶蛋白电泳及电洗脱制备目的蛋白,免疫家兔。酶联免疫吸咐试验(ELISA)和Westernblot检测所制备抗体的滴度和免疫原性。结果利用GST或T7-tag序列在其N端融合,使MR-1在大肠杆菌BL21-CodonPlus(DE3)-RIL得到表达。利用所表达获得的MR-1-T融合蛋白,制备了针对此蛋白的多克隆抗体。ELISA检测所制备抗体滴度达到1∶105,Westernblot显示所制备的多克隆抗体可用于检测天然细胞中的MR-1蛋白。结论MR-1蛋白需在N端与GST或T7-tag序列融合方可实现表达。利用在大肠杆菌表达纯化的蛋白所制备的抗体可用于MR-1生物学功能的研究。 展开更多
关键词 人肌纤生成调节因子1 融合表达 多克隆抗体
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转录因子XBP1的融合表达、纯化及多克隆抗体的制备 被引量:6
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作者 刘雨飞 丁丽华 +5 位作者 郝春芳 方言 赵福弟 黄翠芬 杨晓 叶棋浓 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2004年第6期762-767,共6页
人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82~ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pG... 人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82~ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pGEX KG中 ,构建成重组质粒pGST XBP 1(82~ 14 7位氨基酸 ) .将该重组质粒转化E .coliDH5α后 ,表达GST XBP 1(82~ 14 7位氨基酸 )融合蛋白 ,经谷胱甘肽 Sepharose 4B亲和层析获得纯化的融合蛋白 .用此融合蛋白免疫家兔制备多克隆抗体 .利用制备的抗体分别用Western印迹和免疫细胞化学检测XBP 1的 2种剪切形式在哺乳动物细胞中的表达 .结果表明 ,该抗体对XBP 1的 2种剪切形式均具有反应原性 ,效价高 ,特异性好 ,可以用于进一步研究XBP 展开更多
关键词 人X盒结合蛋白1(XBP-1) 融合表达 多克隆抗体
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P277多肽融合热休克蛋白65提高抗1型糖尿病的作用 被引量:4
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作者 朱爱华 鲁勇 +3 位作者 金亮 吴洁 李泰明 刘景晶 《生物工程学报》 CAS CSCD 北大核心 2008年第4期640-645,共6页
为了提高P277肽抗1型糖尿病的作用,把P277肽融合在卡介苗热休克蛋白65的C端,构建了pET28a- HSP65-P277高效表达载体,在大肠杆菌中高效可溶性表达。利用硫酸铵分级沉淀、阴离子交换柱层析分离纯化了融合蛋白HSP65-P277。使用HSP65-P277... 为了提高P277肽抗1型糖尿病的作用,把P277肽融合在卡介苗热休克蛋白65的C端,构建了pET28a- HSP65-P277高效表达载体,在大肠杆菌中高效可溶性表达。利用硫酸铵分级沉淀、阴离子交换柱层析分离纯化了融合蛋白HSP65-P277。使用HSP65-P277在没有任何佐剂存在的情况下免疫非肥胖性糖尿病(NOD)小鼠,通过三次腹腔注射,每月收集被免疫动物的血清,血糖浓度用自动生化分析仪测定。结果显示HSP65-P277免疫组小鼠血糖平均值及糖尿病的累积发病率和其余组相比均有显著差异(P<0.01),融合蛋白HSP65-P277抗NOD小鼠糖尿病的作用显著高于单独的P277和HSP65。为进一步开发能用于临床的1型糖尿病疫苗提供了良好的设计思路,HSP65-P277极有可能进一步发展成为新的抗Ⅰ型糖尿病的疫苗。 展开更多
关键词 热休克蛋白 1型糖尿病 疫苗 融合蛋白 血糖
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中国株HIV-1结构蛋白基因gag与gp120在巴斯德毕赤酵母中的融合表达 被引量:3
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作者 江文正 金宁一 +2 位作者 王宏伟 张应玖 金洪涛 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2002年第5期426-428,共3页
目的 将中国流行株B亚型HIV 1结构蛋白基因gag和 gp12 0在巴斯德毕赤酵母中进行融合表达。 方法 以酵母分泌型表达质粒 pPIC9为载体 ,构建含HIV 1gag和gp12 0嵌合基因的重组酵母表达质粒pPICGP。用SacI将pPICGP线性化后 ,电转化巴斯... 目的 将中国流行株B亚型HIV 1结构蛋白基因gag和 gp12 0在巴斯德毕赤酵母中进行融合表达。 方法 以酵母分泌型表达质粒 pPIC9为载体 ,构建含HIV 1gag和gp12 0嵌合基因的重组酵母表达质粒pPICGP。用SacI将pPICGP线性化后 ,电转化巴斯德毕赤酵母GS115 ,用PCR的方法鉴定阳性酵母转化子。阳性转化子在巴斯德毕赤酵母中用甲醇进行诱导表达 ,表达产物以SDS PAGE和West ernblot进行分析 ,并对阳性菌株的遗传稳定性进行研究。结果  12个酵母转化子中共筛选到了 8个阳性酵母转化子 ,其整合率为 6 6 .7%。SDS PAGE和Westernblot分析显示 ,在相对分子质量 (Mr)为 5 70 0 0处有 1条特异蛋白带 ,且能与抗p2 4单抗 (mAb)和抗 gp12 0mAb发生反应。酵母转化子在YPD培养基上传代 10次后 ,其外源基因未丢失。结论 在巴斯德毕赤酵母中成功地表达了HIV 1gag gp12 0嵌合基因 ,且表达蛋白具有特异性 ,但其Mr较预计计算的值要小 ,说明嵌合基因中的 gp12 展开更多
关键词 HIV-1 结构蛋白 毕赤巴斯德酵母 融合表达 人工型 免疫缺陷病毒
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