A Primary Study of Infection Mechanism of Hepatitis E Virus on Hepatocytes

戊型肝炎病毒（HEV）不稳定易降解，难以从载毒标本中分离，而迄今为止仍没有有效的细胞培养模型，这很大程度上限制了HEV感染致病机制的研究。近年来，随着对HEV研究的深入，较好模拟了HEV病毒衣壳表面结构的重组蛋白为研究HEV与宿主细胞相互作用提供了一条新的途径。HEV衣壳由单一蛋白（pORF2）组成，大量研究表明pORF2的重组表达片段p239（aa368－606）很好地模拟了HEV的免疫优势中和表位。p239对HEV与原代肝细胞、HepG2细胞的吸附阻断，p239对多株细胞系具有与HEV相似的嗜性，以及多株HEV特异单抗对p239与HepG2细胞结合的特异阻断，强有力地提示p239同时也很好...Hepatitis E Virus [HEV] has emerged to be a dominant cause of acute hepatitis, replacing hepatitis A virus in some areas and comparable in others. Research into pathogenesis and epidemiology of infection caused by this virus has been severely hampered, because the virus could not be efficiently propagated in cell culture. Previous studies suggest that p239, a recombinant protein specified by ORF...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：B20032601

Distribution of Fine Roots in a Mixed Cunninghamia lanceolata-Tsoongiodendron odorum Plantation

对27年生混交比例为2行杉木和1行观光木的混交林和杉木纯林群落细根分布的研究表明,杉木和观光木行间的杉木细根密度虽比杉木与杉木行间的低8.5％,但观光木细根密度则高152.09％,其细根总密度比杉木与杉木行间的大10.43％。混交林中杉木各径级活细根密度呈单峰型分布,均以5-10cm土层最大,而观光木各径级活细根主要分布在0-10cm土层内。纯林杉木各径级活细根密度亦基本呈单峰型分布,但峰值出现在10-20cm或20-30cm土层。不同树种不同径级死细根的分布均与其各自的活细根分布相似。混交林中灌木细根密度在30-40cm的土层最大,而纯林中的灌木细根集中于0-10cm的表土层;混交林和纯林中的草本细根均集中在0-5cm土层。与纯林的相比,混交林中杉木细根主要分布的土层明显上移,表层土壤细根所占比重增大,有利于更好利用土壤养分和提高群落生产力。Fine roots were measured by soil core sampler in a mixed plantation of 27-year-old Cunninghamia lanceolata (C) and Tsoogiodeudron odorum (T) in Fujian Province. Fine root density of C at interrow between C and T was lower by 8.5% than that between C and C, but fine root denstiy of T and total fine root density were higher by 152.09% and 10.43%, respectively. High density of living fine roots with different thickness of C in mixed plantation appeared at 5-10 cm soil depth, whereas that of T at 0-10 cm. Compared to mixed plantation, living fine root density in pure C plantation appeared at 10 -30 cm of soil depth. The distribution of dead fine roots had the same pattern. In mixed plantation, upward trend of soil layer with maximum fine roots of C was obvious, showing that mixed plantation had an advantage over pure plantation in nutrient absorption.中国博士后科研基金项目（1999-10）;;福建省科委重大基础研究项目（2000-F-004）;; 高等学校骨干教师资助计划项目;;福建省自然科学基金项目（B0110025

HepG2细胞中与戊型肝炎病毒衣壳蛋白相互作用蛋白的初步研究

利用重组戊型肝炎病毒(HEV)衣壳蛋白片段p239(368~606 aa)形成的类病毒颗粒作为亲和层析诱饵蛋白,HepG2为细胞模型,筛选与p239类病毒颗粒特异性相互作用蛋白。经过二维电泳分离,MALDI-TOF-MS分析鉴定得到GRP78/Bip,HSP90,alpha-tubulin及P43四个与p239有相互作用的候选蛋白。GRP78/Bip为热休克蛋白家族成员,体外免疫共沉淀实验结果证实,其与p239有特异性的结合。此研究结果为深入研究HEV的感染过程如吸附、入胞,以及HEV的致病机理提供了有益线索

重组痘苗病毒对不同哺乳动物细胞株的感染效率及EGFP表达水平研究

研究重组痘苗病毒对不同哺乳动物细胞的感染效率及表达水平,可为痘苗病毒表达系统宿主细胞的正确选择提供依据.本研究利用重组绿色荧光蛋白基因的痘苗病毒WR-EGFP同时感染不同的哺乳动物细胞株,利用流式细胞仪检测EGFP的表达强度.共使用20种哺乳动物细胞株,其中10种人类组织细胞,2种猴组织细胞,8种小鼠组织细胞.结果表明,重组痘苗病毒WR-EGFP对鼠细胞系BHK21和人细胞系A-549的感染效率和表达效率最佳;整体看,痘苗病毒对多数灵长类动物细胞的感染效率和表达效率优于鼠细胞;对贴壁细胞的感染效率和表达效率明显优于悬浮细胞;但没有特别的组织偏嗜性

戊型肝炎病毒抗体双抗原夹心法ELISA的建立与初步应用

目的　建立抗戊型肝炎病毒 (HEV)抗体检测的双抗原夹心ELISA(DS -ELISA) ,并应用于多种动物血清的检测。方法　将大肠杆菌表达的一段HEVORF2区重组抗原分别包被微孔板和进行辣根过氧化物酶 (HRP)标记 ,利用 5份阳性血清和 4 0份阴性血清建立双抗原夹心ELISA ;用 4 0 0份义务献血员血清比较双抗原夹心ELISA与间接法IgG抗体ELISA的符合情况 ;用 3只HEV感染猴系列血清比较双抗原夹心ELISA试剂和Genelabs公司HEVIgG试剂 ;用双抗原夹心ELISA试剂检测新疆地区的部分牛、绵羊、山羊、猪血清和上海地区的部分鸡血清中的HEV抗体。结果　建立了检测HEV抗体的双抗原夹心ELISA方法 ,对 4 0 0份义务献血员血清的检测表明其与间接法IgG抗体ELISA试剂的符合情况良好 ,并且有更高的s/co比值 ;与Genelabs公司HEVIgG试剂的比较表明双抗原夹心ELISA试剂的检出更早 ,尤其是持续时间及强度明显优于Genelabs试剂 ;在所检测的各种动物中均发现了HEV抗体 ,其中猪抗体的阳性率最高 ,表明双抗原夹心ELISA试剂可同时用于不同动物的抗HEV抗体检测。结论　利用大肠杆菌表达的HEV重组抗原建立了双抗原夹心法ELISA ,并可同时用于不同动物的抗HEV抗体检测

Development and Application of a Novel Neutralization Test for Echovirus 25

目的:建立一种新型的快速、高通量的埃可病毒25型(ECHO25)中和抗体检测方法,并初步评价其在ECHO25中和抗体筛选和血清流行病学调查中的应用价值。方法:应用免疫荧光方法筛选ECHO25高亲和性抗体并将其作为检测单抗,结合酶联免疫斑点检测技术(ELISPOT)建立ECHO25中和抗体检测方法;使用不同效价的血清评价该方法的准确性;采用所建立的中和方法对ECHO25单克隆抗体、临床血清样品进行检测。结果:建立了快速检测ECHO25中和抗体的Nt-ELISPOT方法,以ECHO25单克隆抗体5B9作为检测抗体;相比经典的中和实验方法 Nt-CPE,该方法可显著缩短检测时间(从5~7 d缩短至1 d以内),检测结果具有较好的一致性;采用所建立的Nt-ELISPOT方法首次筛选获得3株对ECHO25具有较好中和能力的单克隆抗体;临床血清样品检测结果显示厦门地区可能存在ECHO25的流行。结论:该方法可以应用于中和抗体筛选和血清学的临床辅助诊断,为ECHO25的防治研究提供支持。Objective: To establish a rapid and high-throughput neutralization test for echovirus 25(ECHO25),and evaluate its application in neutralizing antibody screening and seroepidemiological surveys. Methods: Immuno-fluorescence assay was applied to screen a high affinity antibody, which was used as the detection antibody forECHO25, and a rapid neutralization test was established based on enzyme- linked immunospot assay(Nt-ELISPOT). The accuracy of this method was evaluated by detecting serum samples with different titer. Monoclonalantibodies against ECHO25 and clinical serum samples were detected via the established neutralization test. Results: A rapid method to detect neutralizing antibody against ECHO25 was established and an anti-ECHO25 anti-body, 5B9, was used as the detection antibody. The detection period could be shortened significantly comparedwith the classical neutralization test(Nt- CPE)(from five to seven days to less than one day), and the Nt-ELISPOT had good consistency with the Nt- CPE. Meanwhile, three neutralizing antibodies for ECHO25 werescreened firstly by this method. The detection results of clinical serum samples showed that infection of ECHO25 might be popular in Xiamen. Conclusion: This method can be used in neutralizing antibody screening and seroepi-demiological surveys, and it may provide support for the control of ECHO25.国家自然科学基金(81371817,81401669

荧光素标记检测乙型肝炎病毒表面抗原特异性杀伤性T淋巴细胞方法的探讨

用绿色荧光前体化合物CalceinAM标记靶细胞,经过与杀伤性T淋巴细胞(CTL)效应细胞数小时的孵育,通过分析培养上清中的荧光强度检测CTL效应。通过对一系列标记条件的研究:不同浓度CalceinAM标记靶细胞、不同洗涤缓冲液、不同标记细胞浓度、不同洗涤次数、不同裂解液配方,确定CalceinAM荧光素标记法的最佳条件。用该方法检测乙型肝炎病毒表面抗原的DNA疫苗免疫Balb/c小鼠对P815S细胞的CTL效应,E/T比为10时,杀伤效率接近饱和,达到65%。通过荧光显微镜直接观察杀伤后的P815S细胞,细胞破裂程度与计算出的杀伤效率有一定相关性

人肝细胞内戊型肝炎病毒结合蛋白的酵母双杂交筛选

为进一步深入研究戊型肝炎病毒(HEV)感染机制以及致病机理,用酵母双杂交系统从人肝细胞cDNA文库中筛选与戊型肝炎病毒衣壳蛋白E2相互作用的蛋白,核酸序列分析及同源性检索表明,4个克隆与E2相互作用,其中一个克隆与P38IP高度同源,细胞免疫共沉淀实验结果显示:在哺乳动物细胞水平仍能够检测到E2与P38IP片段的特异的相互作用

戊型肝炎病毒细胞吸附模型的建立及病毒吸附区域初步研究

E.coli中表达的HEV衣壳蛋白片段P239(aa368~606)形成的类病毒颗粒与戊肝患者恢复期血清及中和单抗具有良好的反应性,较好地模拟了天然HEV病毒颗粒的表面空间结构。利用P239吸附HepG2细胞的模型来模拟HEV对宿主细胞的吸附,多株中和单抗对吸附的阻断验证了吸附的特异性。P239与多株传代细胞系的吸附结果则表明了这种特异性吸附的细胞选择性。对阻断P239吸附的线性单抗进行定位,初步确定了P239与细胞相互作用的区域:ORF2上的aa423~443很可能和病毒上的细胞膜受体结合部位非常靠近,或可能直接参与构成了病毒与细胞特异性识别的表位。此本研究为进一步研究HEV与宿主细胞的相互作用提供一定的线索

一个包含HCVIRES的高效真核双顺反子表达载体的构建

In this paper, a new eukaryotic bi-cistronic expression vector containing Hepatitis C Virus(HCV) internal ribosome entry site (IRES) expressing two foreign genes from one mRNA was constructed. The sequence starting from the 5' untranslated region of 18nt to 32nt in HCV core coding region was cloned and then the encephalomyocarditis virus (ECMV) IRES sequence in the commercial vector pIRES was substituted to construct a new vector pCVIR. Green fluorescent protein (GFP) and Hepatitis B virus surface antigen (HBsAg) coding genes were inserted up-stream and down-stream of IRES sequence. The fluorescence intensity of GFP and HBsAg were determined by flow cytometry and ELISA respectively., thus, the expression efficiency of the two vectors, pCVIR and pIRES could be compared. The experimental results showed that the vector pCVIR could translate the GFP and HBsAg genes down-stream of its HCV IRES sequence more efficiently without impairing the expression of genes up-stream of IRES sequence than the vector pIRES.It is..