Structural basis for the dimerization, neutralizing epitope and genotype specificity of hepatitis E virus capsid protein

戊型肝炎是最主要病毒性肝炎之一，由戊型肝炎病毒（HEV）感染导致，常引起大规模爆发流行。随着研究的深入，戊肝作为公共卫生问题和导致的临床疾病的危害性越来越突出。近年来大量证据表明戊肝同时是一种人兽共患疾病，猪是HEV的最主要动物宿主和人类戊肝的重要传染源。 由于缺乏有效的细胞培养模型或小动物模型，HEV感染及复制过程的分子机制一直未被阐明，HEV如何选择宿主、多种基因型却呈现单一的血清型等问题亦悬而未决。HEVORF2编码的结构蛋白组装成HEV的衣壳，与病毒的吸附、穿入、致病性、宿主嗜性和病毒基因型及诱导宿主的免疫应答等作用紧密相关，开展对HEV衣壳蛋白的结构特征、重要免疫表位和宿主细胞受体...Hepatitis E (HE) is one of the major viral hepatitis caused by infection with the hepatitis E virus (HEV), and often leads to outbreaks. With the increasing study, the threats to public health and clinical presentation of HE have been shown to be much broader than previously believed. Recent studies have confirmed that HE is a zoonotic disease and swine is a principal reservoir of HEV that infects...学位：理学博士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：2162007015383

Fibroblast-like synoviocytes from adjuvant arthritis rat were transfected with plasmid over-expressed TIPE2 and its related analysis

目的:通过TIPE2表达质粒转染大鼠佐剂型关节炎(AA)成纤维样滑膜细胞(flS),体外研究AA的发病中TIPE2对dr5介导细胞凋亡的作用。方法:应用MIgr1/TIPE2+/+表达质粒,经脂质体法导入AA-flS细胞中,转染72小时后,荧光显微镜检测质粒自带gfP基因的表达情况确认转染效率,以未作处理的AA-flS为对照组,用半定量逆转录聚合酶链反应(rT-PCr)检测TIPE2 MrnA的表达,用WESTErn blOT法检测TIPE2蛋白的表达变化,MTT法以及流式细胞术检测抗dr5功能性单链抗体zf1对两组细胞生长的影响。结果:成功获得荧光强度90%的TIPE2过表达AA-flS细胞,TIPE2 MrnA及蛋白表达显著提高。抗dr5单链抗体zf1对TIPE2+/+的flS组细胞具有明显生长抑制及凋亡诱导作用,与不作处理的flS组相比差异显著。结论:TIPE2+/+表达质粒明显提升AA-flS的TIPE2蛋白表达水平,TIPE2对dr5介导细胞凋亡有重要作用。Objective: Fibroblast-like synoviocytes from adjuvant arthritis( AA) rat were transfected with plasmid over-ex- pressed TIPE2+ / +,then apoptosis mediated by TIPE2 on DR5 and its pathological role in adjuvant arthritis( AA) were studied in vitro.Methods: The plasmid of MIGR1 / TIPE2+ / +were transfected to AA-FLS by liposome,after 72 h,the expression of GFP gene that plasmid owned was identified by fluorescence microscope in confirm transfection efficiency,TIPE2 mRNA expression was detected by semi-quantitative reverse transcription polymerase chain reaction( RT-PCR),TIPE2 protein expression of AA-FLS were detected by Western blot.The growth resistance of cells mediated by ZF1( DR5 single chain antibody) was detected by MTT method and flow cy- tometry.Results: Plasmid over-expressed TIPE2+ / +were transfected to AA-FLS successfully as the fluorescence intensity reached 90%,TIPE2 mRNA and protein expression were significantly improved.Furthermore,the growth inhibition and apoptosis induction effect on FLS / TIPE2+ / +were significant,compared with FLS without processing.Conclusion: TIPE2 expression level in AA-FLS are significantly improved by transfecting plasmid expressed TIPE2+ / +,TIPE2 plays an important pathological role in apoptosis mediated by DR5.国家自然科学项目基金(81072472); 福建省自然科学项目基金(2012J01416)资

Targeting Nuclear Receptors with Marine Natural Products

National Basic Research Program of China (973 Program) [2012CB910104]; National Natural Science Foundation of China [31200571, 31270776]Nuclear receptors (NRs) are important pharmaceutical targets because they are key regulators of many metabolic and inflammatory diseases, including diabetes, dyslipidemia, cirrhosis, and fibrosis. As ligands play a pivotal role in modulating nuclear receptor activity, the discovery of novel ligands for nuclear receptors represents an interesting and promising therapeutic approach. The search for novel NR agonists and antagonists with enhanced selectivities prompted the exploration of the extraordinary chemical diversity associated with natural products. Recent studies involving nuclear receptors have disclosed a number of natural products as nuclear receptor ligands, serving to re-emphasize the translational possibilities of natural products in drug discovery. In this review, the natural ligands of nuclear receptors will be described with an emphasis on their mechanisms of action and their therapeutic potentials, as well as on strategies to determine potential marine natural products as nuclear receptor modulators

菖郁逍遥方治疗慢性乙型肝炎伴抑郁症的临床疗效及作用机制探讨

目的观察菖郁逍遥方治疗慢性乙型肝炎（chronic hepatitis B,CHB）伴抑郁症的临床疗效,并探讨其作用机制。方法 90例慢性CHB伴抑郁症患者,采用随机数字表法分为治疗组45例,对照组45例。在恩替卡韦治疗的基础上,对照组服用氟哌噻吨美利曲辛片;治疗组服用中药复方菖郁逍遥方,疗程12周。以汉密尔顿抑郁量表17项(Hamilton depression scale,HAMD17)评分观察治疗前、治疗中（治疗4周）、治疗后（治疗12周）两组患者的抑郁状态变化;以副反应量表（treatment emergent symptom scale,TESS）观察两组治疗后副反应;观察治疗前后血清ALT活性、乙型肝炎病毒DNA（hepatitis B virus DNA,HBV DNA）定量、5-羟色胺（5-hydroxytryptamine, 5-HT）、去甲肾腺上素（norepinephrine, NE）、脑源性神经营养因子（brain derived neurotrophic factor,BDNF）水平及内源性大麻素:N-花生四烯酸氨基乙醇（Arachidonoylethanolamine,AEA）、2-花生四烯酸甘油（2-arachidonoylglycerol,2-AG）及其受体（cannabinoid receptor,CBR）1、2指标变化。结果两组治疗中、治疗后的HAMD17评分较治疗前均明显降低（P0.05）。结论菖郁逍遥方具有显著改善CHB患者抑郁状态的临床疗效,作用机制与通过调控内源性大麻素水平来升高单胺类神经递质、BDNF相关。国家自然科学基金资助项目(No.81503529,No.81673660,No.81873242);;福建省科技计划引导性项目(No.2015D0007,No.2016D012);;厦门市科技计划项目(No.3502Z20174028);;厦门市重要重大疾病联合攻关项目(No.3502Z20179047);;福建省卫生计生医学创新科研人才培养项目(No.2018-CXB-28

Construction and identification of interference plasmid targeting on TNFAIP8

目的:构建并筛选出干扰效率最佳的TnfAIP8-SHrnA-P SIrEn-rETrO Q干扰质粒。方法:通过生物软件选择3个TnfAIP8基因干扰位点,构建干扰质粒并测序验证,将干扰质粒及对照质粒分别转染至A549细胞,通过rT-PCr、WESTErn blOT检测干扰效率。结果:经rT-PCr和WESTErn-blOT证实TnfAIP8-SHrnA-P SIrEn-rETrO Q干扰质粒能有效干扰并抑制细胞内TnfAIP8基因的表达,通过流式检测发现降低TnVAIP8表达可以提高细胞对A dr5SC fV诱导凋亡的敏感性。结论:成功构建和设计了对TnfAIP8基因具有显著干扰效率的干扰质粒,为进一步研究TnfAIP8基因的功能奠定了基础。Objective: To construct and screen the high efficiency interference plasmid of TFAIP8-shRNA-p SIRENRetro Q.Methods: Selected and synthesized three Target Sequence of TNFAIP8 shRNA1,TNFAIP8 shRNA2,TNFAIP8 shRNA3,and construct the TNFAIP8 interference plasmid.Transfection TNFAIP8-shRNA-p SIREN-Retro Q interference plasmid to A549 cells.Filter out the highest interference efficiency plasmid by detecting the mRNA and protein levels using RT-PCR and Western blot methods.Results: We successfully design and built three TNFAIP8-shRNA-p SIREN-Retro Q interference plasmids,and screen out the highest efficiency interference plasmid.Conclusion: Three interference plasmids targeting the TNFAIP8 gene have been constructed successfully and provide a useful tool for studying the function of TNFAIP8.国家自然科学基金项目(81272720); 福建省卫计委医学创新课题(2014-CXB-43); 厦门市科技计划项目(3502Z2083008)资

慢性乙型肝炎患者肝组织HBcAg表达模式与中医证型的关系

目的:观察慢性乙型肝炎(CHB)患者肝组织乙型肝炎核心抗原(HBcAg)表达模式与中医证型及肝组织炎症、纤维化等指标的关系并进一步探讨相应的机制。方法:选择CHB患者556例,均进行中医辨证分型及肝组织活检,测定肝组织HBcAg表达模式、炎症、纤维化程度及ALT、AST、HBV DNA水平。结果:(1)556例患者按中医辨证标准分为6组:瘀血阻络证44例、肝肾阴虚证42例、肝郁气滞证48例、肝郁脾虚证282例、湿热蕴结证140例、脾肾阳虚证0例;按HBcAg表达模式分HBcAg阴性组42例、胞浆型HBcAg组186例、胞浆优势型HBcAg组290例、浆核各半型HBcAg组38例。(2)经Spearman秩相关分析,肝组织HBcAg表达模式与肝组织炎症活动度、纤维化程度均呈负相关;经Fisher确切概率检验,肝组织HBcAg表达模式与中医证型分布具有相关性。结论:肝组织HBcAg表达模式与中医证型、肝组织炎症、纤维化程度均有相关性;HBcAg阴性组、胞浆型HBcAg组及肝郁脾虚证、湿热蕴结证多处于慢性乙型肝炎活动期,正气未伤,免疫功能较强,为抗病毒治疗的较佳时机。国家自然科学基金项目(No.81503529,No.81673660);;福建省科技计划引导性项目(No.2015D007,No.2016D012);;厦门市科技惠民项目(No.3502Z20174028);;厦门市重要重大疾病联合攻关项目(No.3502Z20179047);;福建中医药大学基金项目(No.XB2016080)~

分子对接预测H5亚型禽流感病毒的广谱中和表位

H5N1禽流感病毒已经在亚洲、欧洲和非洲广泛传播,造成了巨大损失.最近我们鉴定出一株对多种来源的H5N1代表株均有良好中和活性的、识别H5亚型血凝素(hemagglutinin,HA)的广谱单克隆抗体8H5,它对寻找克服禽流感高变性的广谱治疗性抗体、疫苗和药物具有重要价值.本研究应用分子模拟技术,采用"典范结构"方法对8H5抗体Fab片段进行结构模建,并通过能量分析、SAS值分析、"拉曼强传图"检验、profile-3D分析等理论验证,获得较为合理的8H5Fab的三维空间结构.8H5Fab与3种HA蛋白晶体结构的分子对接结果表明,8H5抗体与HA蛋白的作用模式与HA宿主来源无关,但与HA结构的亚型相似性相关.综合抗原同源比对结果,推测8H5抗体识别的广谱中和表位是由HA上的Asp68,Asn72,Glu112,Lys113,Ile114,Pro118,Ser120,Tyr137,Tyr252不连续氨基酸残基组成的构象表位.这一模型将为H5亚型禽流感病毒广谱疫苗和治疗性药物的分子设计提供依据

大肠杆菌来源的人乳头瘤病毒11型病毒样颗粒的制备及其免疫原性

【目的】利用大肠杆菌表达系统制备人乳头瘤病毒11型病毒样颗粒(HPV11VLPs),并对其免疫原性和所诱导中和抗体的型交叉反应性进行研究。【方法】在大肠杆菌ER2566中非融合表达HPV11-L1蛋白,并通过离子交换层析,疏水相互作用层析其进行纯化。纯化后的HPV11-L1经体外组装形成病毒样颗粒,通过动态光散射,透射电镜检测其形态,并通过多种HPV型别假病毒中和实验评价HPV11VLPs的免疫原性及型交叉反应性。【结果】HPV11-L1蛋白在大肠杆菌中可以以可溶形式表达。经过硫酸铵沉淀、离子交换和疏水相互作用色谱纯化,目的蛋白纯度能够达到95%以上。纯化的HPV11-L1蛋白去除还原剂DTT后,能够在体外自发组装成为直径约50nm、与天然病毒颗粒形态高度相似的VLPs。动物实验结果显示,该病毒样颗粒在小鼠体内的中和抗体半数有效剂量(ED50)为0.031μg,在小鼠体内可诱导高达106的中和抗体滴度,这些中和抗体与HPV6有较好的交叉反应,与HPV18有一定的交叉反应,而与HPV16没有明显的交叉反应,这一结果与分子进化树的分析相一致。【结论】本研究利用原核表达系统获得了具有较高免疫原性的HPV11VLPs,为HPV11预防性疫苗的研制奠定了良好的基础

小学语文作文生活化教学策略探讨

作文能够让学生提升能力的同时，也能够让其有更好的情感认知和人文情怀。小学语文作文教学对教师来讲一直是一大挑战，要让小学生感受到作文学习内容的趣味性，又要对其学习与成长发展有益。生活化的语文作文教学也就应运而生，将生活与小学生的写作相结合，是非常好的教学方式之一。</jats:p