Broad-Spectrum Detection and Identification of Foodborne Pathogens with Multicolor Real-time PCR

快速、有效、高通量地对食源性致病菌进行检测与识别对于疾病预防与控制十分重要。食源性致病菌多种多样，如何从一系列怀疑对象中确定特定的病原已成为亟待解决的重要课题。本论文综合运用实时PCR、多色组合探针编码（MCPC）技术和相同标签辅助-无引物二聚体（HAND）系统，致力于对食源性致病菌的广谱检测与识别。 第一章，综述食源性致病菌的检测现状，介绍传统检测方法、免疫学检测方法和分子生物学检测方法的应用现状及发展趋势。针对目前实时PCR存在检测通量偏低的问题，分析其中的技术瓶颈，提出采用多色组合探针编码技术的思路。 第二章，通过改良分子信标-多重荧光PCR的方法实现食源性致病菌的广谱检测。本章选择...Foodborne bacterial pathogens are recognized as one of the major etiological agents that cause foodborne disease. The availability of rapid, specific and high throughput assays to detect the presence or absence, or even the degree of contamination of pathogens, has become increasingly important for the food industry. This dissertation describes three solutions for the broad-spectrum detection and ...学位：理学硕士院系专业：生命科学学院生物医学科学系_生物化学与分子生物学学号：20042609

The preparation and tumor cell apoptosis-inducing activity assay of anti-human DR5 single-chain antibody

作者简介: 张佳锴,男,硕士研究生,主要从事肿瘤免疫学研究, Tel: 0592-2180587，E-mail: baroce@ 163.com; 庄国洪，通信作者，E-mail: [email protected]。[中文摘要]目的构建、表达、纯化抗人死亡受体5(DR5)单链抗体,并检测其诱导肿瘤细胞凋亡的活性。方法采用RT-PCR获取鼠源性抗人DR5单克隆抗体重链和轻链可变区基因序列,以一柔性连接肽连接二者,转入表达载体,以大肠杆菌表达融合蛋白,亲和色谱纯化后用MTT实验和凋亡检测试剂盒检测其凋亡诱导活性。结果获得的序列经比对为抗体重链和轻链可变区基因,表达纯化后的重组蛋白具有接近完整抗体的肿瘤细胞凋亡诱导活性。结论抗人DR5 scFv可作为诱导肿瘤细胞凋亡的候选药物,为肿瘤免疫学研究提供材料。[英文摘要]Purpose To construct，express，purify anti-human DR5 scFv，and to assay its activity of apoptosis induction for tumor cell lines． Methods Variable region sequences of heavy chain and light chain to murine anti-human DR5 monoclonal antibody were acquired by RT-PCR， then they were linked through a flexible linker peptide and was cloned into the expression vector． After being expressed by E．coli and purified using affinity chromatography， the recombinant protein was employed to MTT assay as well as apoptosis assay kit in order to detect its apoptosis-inducing activity． Results The sequences we've got were established as the variable region genes of antibody's heavy and light chain，while the recombinant proteins expressed and purified possessing the apoptosis-inducing activity come near to complete antibody．Conclusion Anti-human DR5 scFv can supply material for tumor immunology research as drug candidate inducing tumor cell apoptosis．福建省自然科学基金资助项目(C0710046

临床药师干预对提高辅助用药应用合理性的作用研究

目的分析临床药师干预对提高辅助用药应用合理性的作用。方法回顾分析2018年1-12月医院普外科、骨外科、内科、产科、妇科等所收治470例患者的辅助用药资料,由医院临床药师对于辅助用药资料进行专项点评以及巡查,分析临床药师干预对提高辅助用药应用合理性的作用。结果经研究统计发现,470例患者的辅助用药医嘱数量为864份,依据《新编药物学》的分类方法统计分析辅助用药情况发现:医院所应用范围最为广泛的药物包括肠内营养乳剂、脂肪乳(10%)氨基酸(15)葡萄糖(20%)注射液、脂肪乳氨基酸(17)葡萄糖(11%)注射液、多种微量元素注射液(Ⅱ)、脂溶性维生素注射液(Ⅱ)、注射用水溶性维生素、匹多莫德颗粒、奥拉西坦注射液、丹参川芎嗪注射液、参芎葡萄糖注射液、丹红注射液、参麦注射液、丹参注射液、依达拉奉注射液以及注射用鼠神经生长因子。依据数据统计发现,不合理用药的情况主要包括超说明书用法用量、适应证不适宜、联合用药不适宜、溶媒选择不适宜、用药疗程延长、药物配伍不当以及使用未注意用药禁忌证。经临床药师干预后,医院不合理用药情况均有所降低,有效提高了辅助用药的合理性。864份辅助用药数量分别来自普外科、骨外科、神经外科、内科、妇科、产科、儿科、新生儿科。结论在临床辅助用药中,对于辅助用药实施审核评价,应保持客观依据,通过对临床药师进行干预,可提高辅助用药应用合理性,有利于辅助用药风险性的降低

抗人死亡受体5单链抗体ZF1对鼠H22肝癌细胞的作用分析

目的:研究抗人死亡受体5单链抗体ZF1对H22肝癌细胞体内外抑制增殖作用。方法:MTT法检测ZF1对H22肝癌细胞体外杀伤作用,流式细胞术检测ZF1诱导H22的凋亡率,建立H22移植瘤模型,随机分为PBS组、ZF1组、EPI组和ZF1/EPI联合组四组。观察肿瘤生长和小鼠体重变化情况。治疗13天后分离肿瘤组织,进行HE染色检查、TUNNEL法检测细胞凋亡。结果:体外实验显示:ZF1可抑制H22细胞的增殖,呈剂量依赖性,抑制率最高为84.5%。体内实验结果显示单独应用ZF1或联合应用ZF1/EPI时,肿瘤增长受到明显抑制。HE染色和TUNNEL分析结果表明ZF1可有效诱导肝癌肿瘤凋亡,ZF1/EPI联合组效果更明显,而对正常肝细胞无毒性。结论:单链抗体ZF1具有良好抑制H22细胞增殖的作用。ZF1和EPI联合应用效果更明显

抗人DR5单链抗体诱导HepG2细胞凋亡的实验研究

目的探究抗人DR5单链抗体(scFv)ZF1对肝癌细胞株HepG-2的凋亡作用及机制。方法 MTT法检测ZF1对HepG-2的细胞毒效应;流式细胞术检测ZF1诱导HepG2的凋亡率;荧光显微镜观察经ANNEXIN-V/PI双染的HepG-2细胞;DNA Ladder检测HepG-2细胞的DNA特点;Western blotting检测Bcl-2、PARP蛋白的表达。结果 MTT结果显示ZF1抑制HepG-2细胞生长呈剂量依赖性,ZF1终浓度分别为0.225、0.45、0.9mg/ml、1.2mg/ml200μl时,HepG-2细胞的生长抑制率分别为28.8%、52.3%、65.3%、89.8%;流式细胞仪检测结果显示,终浓度分别为0.225、0.45、1.2mg/ml2ml作用HepG-2细胞4h,凋亡率分别为32.9%、56%、83.2%;荧光显微镜下可见早期凋亡细胞,细胞膜呈绿色荧光,细胞内有凋亡小体的形成,伴有核结构的变化;DNALadder出现明显的彗星尾状条带;Western blotting检测到ZF1诱导凋亡的细胞内Bcl-2、PARP蛋白表达上调。结论 ZF1可诱导HepG2细胞株凋亡,这种作用与HepG2细胞株表达Bcl-2、PARP蛋白蛋白表达上调相关

Fas胞外区基因的构建、表达、纯化及多克隆抗体的制备

目的构建Fas胞外区(eFas)基因的表达载体,表达纯化重组蛋白,进行多克隆抗体的制备,为进一步功能研究奠定基础。方法通过重叠PCR获得eFas基因的编码序列,构建pET-22b(+)/eFas表达载体,转化大肠杆菌Rosetta-gami,IPTG诱导表达,Ni-NTA柱亲和纯化,SDS-PAGE鉴定重组蛋白的纯度。将纯化的eFas融合蛋白免疫新西兰白兔制备多克隆抗体,通过ELISA方法检测多克隆抗体的效价。结果获得了eFas的编码序列与表达载体,目的蛋白主要在包涵体中表达,表达量占菌体总蛋白的30%以上,纯化的重组蛋白纯度达95%以上。结论eFas融合蛋白基因的构建、表达、纯化以及多克隆抗体的制备,为进一步研究Fas提供了材料

Characterization of single chain antibody against DR5

目的探究抗人死亡受体5(dr5)单链抗体(Adr5SCfV)的特异性。方法 ElISA检测Adr5SCfV的效价、亲和力和表位分析。WESTErn blOTTIng检测单链抗体的特异性。MTT检测Adr5SCfV对结肠癌细胞SW480的生长抑制。结果 ElISA显示抗人死亡受体5单链抗体抗体效价为1.2x104。通过间接ElISA证实Adr5SCfV与Adr5MAb识别dr5胞外段(Edr5,系本实验室自己构建)的同一个位点。Adr5SCfV的亲和力常数为2.12x10-2。Edr5与Adr5SCfV能够特异结合。MTT检测结果显示,Adr5SCfV抑制SW480细胞生长呈剂量依赖性,Adr5SCfV终质量浓度分别为0.225 Mg/Ml、0.45 Mg/Ml、0.9 Mg/Ml、1.2 Mg/Ml。200μl时,SW480细胞的存活率分别为97.7%,70.9%、70.1%、23.6%。结论 Adr5SCfV能与Edr5特异性结合,并有较强的活性。The aim of our study is to characterize the property of aDR5ScFv.We employed ELISA to determine the titer,relative affinity and epitope recognition of aDR5ScFv,while Western blotting to analyze the specificity of aDR5ScFv.We also measured the inhibitory effects of aDR5ScFv on colon cancer cell SW480 growth by MTT.We found that the titer of aDR5ScFv was 1.2×104;aDR5ScFv and aDR5mAb recognized the same epitope on DR5 molecule;and the affinity constant of aDR5ScFv was 2.12×10-2.Furthermore,aDR5ScFv could recognize eDR5 specifically and inhibit the growth of SW480 cells in a dose-dependent manner(aDR5ScFv final concentrations of 0.225,0.45,0.9,1.2 mg/ml correspond to SW480 viabilities of 97.7%,70.9%,70.1%,23.6%).Western blotting showed the binding of aDR5ScFv to DR5 was specific.Thus,we concluded that binding of aDR5ScFv to DR5 is specific and aDR5ScFv had strong activity.福建省自然科学基金(C0710046

Direct Fabrication of Ultrafine Electrospinning Nanofiber

采用装配有疏水铜网的新型喷头研究了超细纳米纤维的制备.静电纺丝实现之前,首先对铜网进行了疏水处理,并将其安装于喷头前端.静电纺丝过程中,聚合物溶液由精密注射泵输送至喷头处.安装于喷头的铜网可将管道内的聚合物溶液分成多股细流从铜网网孔中流出.从铜网网孔流出的溶液细流受电场力作用被拉伸成多股独立射流,并从喷头携带走聚集的正电荷.受铜网表面疏水性和射流间电荷排斥力的影响,从铜网喷射出的多股射流都将保持其独立的轨迹而不会产生聚集.疏水铜网有利于减小纺丝射流的初始直径,并获得均匀的超细纳米纤维.利用新型的电纺丝喷头成功制备了直径20~80 nM的聚氧化乙烯(PEO)和聚乙烯醇(PVA)超细纳米纤维.实验结果表明,超细纳米纤维的直径随着电纺丝溶液浓度的增加而变大.A novel spinneret assembled with Cu grid was presented in this paper to fabricate ultrafine nanofiber directly.Before electrospinning,hydrophobic treatment was performed on the Cu grid,which was then fixed at the front end of spinneret.During electrospinning,the polymer solution was transferred to the spinneret by the precise syringe pump.Through the holes in the Cu grid,polymer solution flow was divided into several smaller ones.The fine liquid flow from each hole of Cu grid was stretched into individual jets by the electric field force,and the liquid jets carried away the positive charges accumulated on the spinneret.Due to the hydrophobic treatment and the charge repulsive force between charged jets,liquid jets emanated from Cu grid kept their own tracks without aggregation.The initial diameter of liquid jet was greatly decreased by the Cu grid after hydrophobic treatment,and the smaller jet led to finer uniform nanofiber.Polyethylene oxide(PEO) and polyvinyl alcohol(PVA) ultrafine nanofiber with the diameter of 20—80 nm were fabricated by this novel spinneret,and the diameter of ultrafine nanofiber increases with the increase of polymer solution concentration.国家自然科学基金重点资助项目(51035002);国家自然科学基金资助项目(50875222

改良分子信标-实时PCR快速检测志贺菌

目的:建立改良分子信标-实时PCR检测志贺菌的快速方法,应用于志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。方法:根据GenBank公布志贺菌ipaH基因的保守序列,设计引物和改良分子信标探针,建立实时PCR-改良分子信标检测体系,应用于对志贺菌食物中毒的快速诊断和门诊肠道致病菌的检测。结果:改良分子信标-实时PCR反应体系DNA灵敏度为93 fgμ/l,菌液灵敏度为64 cfu/m l或2 cfu/PCR反应体系,无交叉反应。此反应体系检测67株志贺菌,均出现特异的荧光信号。对细菌性食物中毒样本等共657份样品进行志贺菌检测,42份志贺菌实时PCR阳性,其中41份志贺菌细菌培养阳性。从样品处理到检测结果仅需2 h~1 d时间。结论:改良分子信标-实时PCR检测体系快速、灵敏度高,特异性强,可用于志贺菌食物中毒的快速诊断,为食源性疾病的分子流行病学调查提供新的检测手段,对于提高肠道门诊的工作效率有重要意义

改良分子信标实时PCR快速检测大肠杆菌O_(157):H_7

目的建立改良分子信标实时PCR快速检测大肠杆菌O157H7的快速方法。方法根据GenBank公布O157H7的rfbE保守序列,设计一对引物和改良分子信标探针,用FAM荧光剂标记探针的5’端,建立改良分子信标实时PCR检测O157H7的反应体系,应用于食物中毒快速诊断和食品微生物检测。结果共检测11种细菌,只有大肠杆菌O157H7有荧光信号,其余10种全无荧光信号,且与其他细菌无交叉反应,DNA灵敏度为64fg,菌液灵敏度为59cfu/ml或2cfu/PCR反应体系。应用改良分子信标实时PCR反应体系检测10株O157H7均出现特异的荧光信号,无干扰。对89份食品样品进行检测,5份O157H7实时PCR阳性,其余样品为阴性,检测仅需2h。5份阳性样本,经传统方法培养,有3份检出大肠杆菌O157H7。结论改良分子信标实时PCR检测体系快速、灵敏度高,特异性强,可用于大肠杆菌O157H7食物中毒的快速诊断和食品微生物检测,为食源性疾病的分子流行病学调查提供新的检测手段