Preparation and Preliminary application of Dextran Quantitative Detection Kit

右旋糖酐（Dextran），又名葡聚糖，由Leuconostocmesenteroides等微生物产生的酶聚合葡萄糖而成或葡萄糖通过化学聚合而成。右旋糖酐广泛应用于医药、食品、化工等领域。在医学领域的应用主要包括：作为血浆体积扩张剂，作为抗凝剂，以及作为渗透压调节剂用于相关的疾病治疗。一般机体组织细胞并不能直接利用右旋糖酐，主要从尿液排出，右旋糖酐使用过量会引起急性肾衰竭、充血性心衰竭、肺水肿及血小板减少等毒副作用。因此，监测血液、尿液的右旋糖酐含量是指导右旋糖酐用量的重要指标，同时右旋糖酐也是诊断血液或尿道微生物感染的依据，另外右旋糖酐的定量监测也是食品工业质控的一个重要指标与技术。本论文研...Dextran is a kind of polymer of macromolecule glucose, and is produced by glucose polymerizing with enzymes generated by microorganisms such as Leuconostoc mesenteroides or by chemical polymerization. Dextran is widely used in medicine, food, chemical industry, etc. The applications of dextran in medical fields mainly include: 1, as the plasma volume expander. 2, as the anticoagulant. 3, as the os...学位：工程硕士院系专业：化学化工学院_工程硕士(化学工程)学号：X201219200

QTL Mapping, Epistasis and Environmental Effects Analysis for Rice Chalkiness Trait

水稻的垩白性状是当前限制中国稻米品质提升的最主要因素.研究垩白形成机理及遗传特性,将有利于提高育种中垩白性状的改良效率.本课题组先前构建了广陆矮; 4号/佳辐占重组自交系(GJ RIL)及遗传图谱.本研究连续2年在上杭县和龙海市两地共种植6季GJ; RILs,据各季垩白性状表型数据进行遗传分析,结合遗传图谱进行QTL定位、上位性分析和环境效应分析.遗传分析发现垩白粒率和垩白度呈偏态分布,推测; 垩白性状受主效基因与微效基因共同影响.QTL定位中,垩白粒率获得3个QTLs,qPGWC2、qPGWC4和qPGWC5,遗传贡献率分别为2.84; %、3.74%和14.09%;垩白度获得3个QTLs,qDEC1、qDEC4和qDEC5,遗传贡献率分别为2.96%、4.88%和7.79%.上; 位性分析中,垩白粒率和垩白度分别获得7对和5对上位性QTLs,贡献率为0.23%~3.55%.RM307~RM518区间内同时检测到垩白粒率和垩; 白度的QTLs,并参与了垩白粒率和垩白度的上位性互作.RM598~RM5140区间内也同时检测到垩白粒率和垩白度的QTLs,也参与了垩白度的上位; 性互作.环境效应分析发现,垩白度的3个QTLs及~eqDEC10和~eqDEC9这对上位性QTLs均与2010年早季龙海种植环境发生显著或极显著; 的互作效应.Rice (Oryza sativa L.) chalkiness is the most important limiting factor; for currently improving rice quality in China. Studying rice chalky; formation mechanism and hereditary character will be helpful for; increasing the efficiency of the improvement of chalky quality in; breeding. A Guanglu'ai No.4/Jiafuzhan recombinant inbred line(GJ RIL); was developed and its genetic map was constructed previously by our; research group. In this study, the genetic analysis of rice chalkiness; was carried out basing on the chalky phenotyping data of GJ RIL from a; total of 6 growth seasons of two different locations, Shanghang county; and Longhai city, in two consecutive years. Then the QTL mapping,; epistasis and environmental effects of chalkiness were studied by using; these phenotying data and the genetic map. The genetic analysis; indicated that percentage of grains with chalkiness (PGWC) and degree of; endosperm chalkiness (DEC) showed skewness distribution, suggesting that; chalkiness trait was affected by both major and minor genes together. In; QTL mapping, three PGWC QTLs, qPGWC2, qPGWC4 and qPGWC5, were detected,; which explained 2.84%, 3.74% and 14.09% of the genetic variation,; respectively. Three DEC QTLs, qDECl, qDEC4 and qDEC5, were mapped, which; explained 2.96%, 4.88% and 7.79% of the genetic variation, respectively.; In QTL epistasis analysis, 7 and 5 pairs of epistasis QTLs for PGWC and; DEC were identified respectively, and their contribution rates ranged; from 0.23% to 3.55%. The RM307~RM518 interval harbored the PGWC and DEC; QTLs, which were involved in epistatic interaction of PGWC and DEC; respectively. The RM598~RM5140 interval also harbored the PGWC and DEC; QTLs, which also participated in epistatic interaction of DEC.; Environmental effect analysis showed the three DEC QTLs (qDECl, qDEC4; and qDEC5) and a pair of epistatic QTLs between ~eqDEC10 and ~eqDEC9 all; exhibited significant or very closely significant interaction effects; under the environmental conditions in early season at Longhai city of; Fujian province in 2010.福建省中青年教师教育科研项目; 厦门大学中央高校科研基本业务

Expression and activity characterization of thrombosis targeting protein hu3D3V_H-tTF to blood vessel of lung cancer

目的制备一种用于肺癌血管靶向栓塞治疗的融合蛋白hu3D3VH-tTF,并鉴定其生物学活性。方法利用重叠PCR技术构建tTF与hu3D3VH的融合基因,克隆至表达载体pET22 b(+),在E.coliBL21(DE3)中表达,镍亲和色谱柱纯化目的蛋白。ELISA检测融合蛋白hu3D3VH组分与肺腺癌细胞A549选择性结合活性,凝血实验和FⅩ活化实验鉴定融合蛋白tTF组分的促凝血活性。结果获得序列正确的hu3D3VH/tTF/pET22 b(+)重组子,融合蛋白在E.coliBL21(DE3)中高效表达。纯化后的融合蛋白与肺腺癌细胞A549具有选择性结合活性,并能活化FⅩ、有效促发血液凝固。结论成功构建hu3D3VH/tTF/pET22 b(+)重组子,hu3D3VH/tTF融合蛋白具有hu3D3VH的选择性结合能力同时具有TF的促凝血活性,为开展选择性肺肿瘤血管血栓性栓塞研究奠定了基础。 【英文摘要】 Purpose To prepare the fusion protein of hu3D3VH-tTF for thrombosis targeting therapy of lung cancer and to analyze its biological activities.Methods Fusion gene hu3D3VH-tTF was constructed by overlap PCR,cloned into expression vector pET22 b(+),and expressed in E.coli BL21(DE3).The fusion protein hu3D3VH-tTF was purified through Nickel-affinity chromatography column.The selective binding activity of the hu3D3VH moiety of fusion protein was analyzed using ELISA and the coagulation activity of the tTF moiety...福建省自然科学基金项目(C0410004);; 厦门大学科技创新项目(XDKJCX20053026

靶向血栓蛋白(RGD)_3-tTF与肿瘤血管标志物α_vβ_3特异性结合能力的研究

背景与目的:研究靶向血栓蛋白(RGD)3-tTF融合蛋白结合肿瘤血管标志物整合素αvβ3的能力,旨在探讨融合蛋白的RGD多肽数量和化学结构与其结合整合素αvβ3能力的关系及其意义。材料与方法:用3个串联的RGD多肽与截短组织因子(truncated tissue factor,tTF)合成融合基因(RGD)3-tTF,表达于大肠杆菌(E.coli)BL21(DE3),用镍柱纯化融合蛋白。通过凝血实验检测融合蛋白tTF组分的活性,运用间接酶联免疫吸附试验(ELISA)分析其特异性结合整合素αvβ3的能力,并与RGD-tTF融合蛋白的活性进行比较。结果:(RGD)3-tTF与RGD-tTF融合蛋白凝血活性相似(F=0.019,P>0.05),但(RGD)3-tTF融合蛋白特异性结合整合素αvβ3的能力明显升高(F=140.17,P<0.01)。当融合蛋白浓度为0.24μmol/L时,(RGD)3-tTF融合蛋白的OD405nm值是RGD-tTF融合蛋白的1.32倍(1.25/0.95)。结论:(RGD)3-tTF融合蛋白带有两个二硫键和3个RGD多肽,保留了组织因子凝血活性的同时,提高了与整合素αvβ3特异性结合的能力,为开展选择性肿瘤血管血栓靶向治疗的动物实验奠定了基础

(RGD)_3-tTF融合蛋白选择性结合结肠癌裸鼠模型肿瘤血管的实验研究

背景与目的:肿瘤血管作为肿瘤分子靶向治疗的靶点受到广泛的关注,近年来有报道称利用抗体(Ab@作为组织因子(TF@胞外区截短组织因子(truncated tissue factor,tTF@的载体,表达的抗体--截短组织因子(Ab-tTF@融合蛋白能够选择性结合肿瘤血管,诱发实体肿瘤组织血管栓塞,导致肿瘤衰退,但是该方法存在一些弊端。本实验旨在研究以精氨酸-甘氨酸-天冬氨酸(GRGDSP,RGD@多肽作为tTF载体所表达的(RGD@3-tTF融合蛋白选择性结合结肠癌裸鼠模型肿瘤血管的能力。方法:用3个串联的RGD多肽与tTF合成融合基因(RGD@3-tTF,表达于大肠埃希菌(Escherichia coli,E.coli@BL21(DE3@,用镍柱纯化融合蛋白。用RGD-tTF融合蛋白作对照,通过凝血实验和凝血因子Ⅹ(FⅩ@活化实验检测(RGD@3-tTF融合蛋白的凝血活性,运用间接酶联免疫吸附试验(ELISA@方法分析其特异性结合肿瘤血管标志物整合素αvβ3的能力。结肠癌裸鼠模型分为3组(每组1只@,肿瘤组织分别用异硫氰酸荧光素(FITC@标记的(RGD@3-tTF、RGD-tTF融合蛋白、tTF进行荧光染色,免疫荧光实验分析融合蛋白在结肠癌裸鼠模型组织的定位。结果:(RGD@3-tTF融合蛋白保留了组织因子的凝血活性,在Ca2+存在时随着融合蛋白浓度的增加,凝血时间相应缩短,浓度为6μmol/L时,凝血时间为(9.96±0.56@min(与对照组比较,P0.05@。同浓度(RGD@3-tTF融合蛋白与整合素αvβ3的结合能力强于RGD-tTF(F=164.81,P<0.01@,当融合蛋白浓度为0.24μmol/L时,(RGD@3-tTF融合蛋白和RGD-tTF融合蛋白的A405nm分别为1.25和0.95。免疫荧光实验显示,(RGD@3-tTF融合蛋白富集于结肠癌裸鼠模型的肿瘤血管。结论:(RGD@3-tTF融合蛋白在保留组织因子凝血活性的同时通过高效、特异地结合肿瘤血管标志物整合素αvβ3,选择性地定位在结肠癌裸鼠模型的肿瘤血管上,为发展tTF作为效应因子的结肠癌分子靶向治疗奠定了基础

抗CD5单链抗体的基因构建、蛋白纯化及活性鉴定

目的构建高表达的抗CD5单链抗体(scFv anti-CD5)工程菌,为开展以抗CD5单链抗体为构件的肿瘤免疫治疗研究奠定基础。方法从Genbank中获得抗CD5单克隆抗体H65的重链可变区(VH)和轻链可变区(VL)的基因序列,用连接肽的编码序列连接,用重叠PCR技术获得目的基因,酶切后插入pET22b(+)构成重组质粒。测序正确的重组质粒转入E.coliBL21(DE3)诱导表达,产物经Ni-NTA柱纯化后复性,流式细胞术和Western blot-ting检测活性。结果获得序列正确的重组质粒,工程菌表达量高,产物复性后具有识别CD5抗原的活性。结论成功构建了高表达scFv anti-CD5的工程菌,为scFv anti-CD5的应用奠定了基础

Studies of preparation and properties of RGD-tTF water-based ferrofluids

目的探讨RGD-tTF水基磁流体通过磁场和RGD多肽在体外对内皮细胞双靶向的功能。方法通过化学沉淀法以柠檬酸钠为表面活性剂制备水基磁流体(MnFe2O4),弱酸改性后与重组的RGD-tTF融合蛋白结合,利用H-600透射电镜观测纳米粒径,以SUQID鉴定磁性,用MTT法、因子X活化检测和流式细胞仪检测RGD-tTF磁流体生物活性。结果成功制备出的水基磁流体能在磷酸盐缓冲液中稳定分布且具有生物兼容性,实验表明RGD-tTF与水基磁流体结合后对RGD和tTF生物活性均无显著影响,并证实在磁场的作用下能实现了水基磁流体对RGD-tTF的定位作用。结论一种具有内皮细胞双靶向功能的RGD-tTF水基磁流体已制备成功。 【英文摘要】 Purpose To study the property of RGD-tTF water-based ferrofluids double targeting EC304 cells in vitro by magnet and RGD peptide.Methods Water-based ferrofluids(MnFe2O4) were prepared by chemical precipitation method using citrate as surfactant, dispersed in weak acid to create surface charges,and coated with recombinant RGD-tTF protein.Proteins coated ferrofluids were characterized by H-600 transmission electron microscopy(TEM)and Superconducting Quantum Interference Device(SQUID),and its biological activi...教育部和厦门大学出国留学人员启动基金资

pJAK2 polypeptide,an antagonist of suppressors of cytokine signaling-1,can enhance the antitumor effect of dendritic cells

目的:探讨细胞因子信号转导抑制因子-1(SuPPrESSOrS Of CyTOkInE SIgnAlIng1,SOCS1)拮抗物PJAk2多肽(氨基酸序列号为1001-1013)参与树突状细胞(dEndrITIC CEllS,dCS)的体外诱导培养后对dCS抗肿瘤作用的影响。方法:采集健康人外周血,离心获得单个核细胞,用重组人粒细胞巨噬细胞集落刺激因子(rECOMbInAnT HuMAn grAnulOCyTE-MACrOPHAgE COlOny STIMulATIng fACTOr,rHgM-CSf)及重组人白细胞介素-4(rECOMbInAnT HuMAn InTErlEukIn-4,rHIl-4)诱导dCS,第5天分为4组:单纯dCS培养(对照)组、抗原负载(lySATE-dCS)组、多肽修饰(PJAk2-dCS)组和抗原与多肽共培养(lySATE+PJAk2-dCS)组,第6天各组加入肿瘤坏死因子-α(TuMOr nECrOSIS fACTOr-AlPHA,Tnf-α)促成熟。倒置显微镜下观察dCS形态;fCM法检测dCS表型;乳酸脱氢酶(lACTATE dEHydrOgEnASE,ldH)细胞毒实验检测各组细胞毒性T淋巴细胞(CyTOTOXIC T lyMPHOCyTE,CTl)对胃癌细胞bgC-823的靶向杀伤作用;ElISA法检测白细胞介素-12(InTErlEukIn-12,Il-12)和γ干扰素(InTErfErOn-γ,Ifn-γ)的水平。结果:与未加入诱导剂组相比,各组均成功诱导出成熟dCS,均高表达Cd80、Cd83、Cd86和人类白细胞dr抗原(HuMAn lEukOCyTE AnTIgEn dr,HlA-dr),但以lySATE+PJAk2-dCS组的表达水平最高。在10:1~30:1的效靶比范围内,CTl杀伤作用与效靶比呈正相关。当效靶比为30:1时,对照组的CTl杀伤率达(19.77±2.34)%,低于其他3组(P<0.01),而lySATE+PJAk2-dCS组较lySATE-dCS组及PJAk2-dCS组都高(P<0.05)。lySATE+PJAk2-dCS组培养上清液中Il-12及Ifn-γ的分泌水平明显高于对照组(P<0.01)。结论:SOCS1拮抗物PJAk2多肽(1001-1013)可增强dCS对胃癌细胞的抗原递呈及特异性抗肿瘤作用。Objective:To investigate the effect of pJAK2 polypeptide,an antagonist of SOCS1(suppressors of cytokine signaling 1),on antitumor effect of in vitro cultivation-induced DCs(dendritic cells).Methods:Peripheral blood was collected from the healthy volunteers,and the PBMCs(peripheral blood mononuclear cells)were isolated.DCs were induced by rhGM-CSF(recombinant human granulocyte-macrophage colony-stimulating factor)and rhIL-4(recombinant human interleukin-4).On the fifth day,DCs were divided into four groups:control group,Lysate-DCs group,pJAK2-DCs group,and Lysate + pJAK2 DCs group.On the sixth day,TNF-α(tumor necrosis factor-alpha)was added into each group.The morphological features of DCs were observed under an inverted microscope;the phenotypes were detected by FCM(flow cytometry);the killing effect of CTLs(cytotoxic T lymphocytes)on gastric cancer BGC-823 cells was evaluated by LDH(lactate dehydrogenase)cytotoxicity test;the concentrations of IL-12(interleukin-12)and IFN-γ(interferon-γ)were detected by ELISA(enzyme-linked immuno sorbent assay).Results:Mature DCs presented typically morphological and phenotypic features;the DCs in Lysate + pJAK2-DCs group had the highest expression levels of CD80,CD83,CD86 and HLA-DR(human leukocyte antigen DR).When the ratio of effectors to target cells ranged from 10:1 to 30:1,the killing activity of CTLs had a positive correlation with the ratio.When the ratio of effectors to target cells was 30:1,the killing activity of CTLs in the control group was(19.77±2.34)%,which was lowest as compared with the other groups(P < 0.01),meanwhile the killing activity of CTLs in Lysate + pJAK2-DCs group was higher than those in Lysate-DCs and pJAK2-DCs groups(P < 0.05).The levels of IL-12 and IFN-γ secretion in Lysate + pJAK2-DCs group were apparently higher than those in the control group(P < 0.01).Conclusion:An antagonist of SOCS1,pJAK2 polypeptide,can enhance the ability of antigen presentation and specific antitumor effect of DCs on gastric cancer cells.南京军区医学科技创新课题资助项目(编号:10MA068); 福建省自然科学基金面上项目(编号:2010D013); 厦门市科技计划创新项目(编号:3502z20104014

Expression and activities analysis of a fusion protein CREKA/tTF

作者简介: 苏毅(19832) ,男,福建人,硕士; 颜江华(19632) ,男, 通信作者,副教授,Tel :059222180587 ,E2mail :jhyan @xmu. edu. cn。目的制备用于靶向肿瘤血管中的凝血血浆蛋白的CREKA/tTF融合蛋白,并鉴定其生物学活性。方法利用PCR技术构建CREKA与tTF的融合基因,克隆至表达载体pET22b(+),在E.coli BL21中表达,镍亲和色谱柱纯化,梯度透析复性。利用凝血时间,结合荧光定量测定等实验在体外鉴定该融合蛋白的活性。结果获得序列正确的CREKA/tTF/pET22b(+)重组子,融合蛋白在E.coli BL21中高效表达。纯化后的融合蛋白具有引起血液凝固的功能,且能与凝血血浆蛋白结合。结论成功构建CREKA/tTF/pET22b(+)重组子,CREKA/tTF融合蛋白具有TF凝血活性及CREKA的结合活性。国家自然科学基金项目(30973485);厦门大学科技创新项目(XDKJCX20053026

人红细胞凝集型单抗的制备与鉴定

目的制备人红细胞凝集型单克隆抗体并进行鉴定。方法以3种方式处理人血红细胞,获得红细胞免疫原,分别免疫Balb/c小鼠。应用杂交瘤技术,取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,以凝集实验筛选分泌凝集型抗体的杂交瘤细胞,继而用有限稀释法克隆3次,并对杂交瘤细胞的稳定性进行测定。通过腹水法制备抗体,亲和层析法纯化抗体,对抗体的特性进行研究。结果 3种方式获得的免疫原均能建立有效的免疫反应,低渗加超声破碎法所获得的小鼠血清抗体效价最高。通过融合获得8株可稳定分泌抗体的杂交瘤细胞,其中,4A1分泌的抗体凝集效价最高。抗体可使4种人血型的红细胞凝集,无种属交叉凝集反应。结论有效制备了针对人红细胞的凝集性单抗,为其之后的应用研究奠定了基础。福建省省属公益类项目(2015R1036-3,2016R1034-2,2017R1036-3);;福建省科技计划(2014Y0082,2014Y0083