Construction of in vitro infection model of human papillomavirus pseudovirion by optimized baculovirus transduction method

HPV感染是常见的性传播疾病，与宫颈癌及泌尿生殖道相关癌症的发生关系密切，严重危害人类健康。开发安全有效的HPV预防疫苗有望消灭或显著降低HPV感染造成的危害，具有十分重大的社会意义。我实验室正在研究开发HPV基因工程疫苗，迫切需要可简便有效对候选疫苗的保护性进行评估的方法。目前的HPV体外感染模型在实验周期、稳定性、滴度等方面存在各自的缺点，而新型实验技术平台的发展为构建更有效的感染模型提供了有利条件。杆状病毒近几年来被发现可作为一种对哺乳动物细胞的新型基因转移载体，相比其它基因转移方法具有许多独特的优势。本论文即探讨了应用重组杆状病毒在哺乳动物细胞中构建HPV假病毒的可行性。为了更有效的获...Human papillomavirus can cause cervical cancer and other related cancers. It is very important to research and develop HPV vaccines. The recombinant HPV vaccine is underdeveloping in our lab. And the simple and efficient method for estimation of the protective ability of the candidate vaccine was needed. However currently available in vitro infection models are technically demanding or relatively ...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：B20012601

水痘带状疱疹病毒皮层蛋白功能的研究进展

水痘带状疱疹病毒(VzV)初次感染可引发水痘并可长期潜伏在宿主神经节内,一旦再激活即可引起带状疱疹.VzV病毒具有疱疹病毒典型结构,从内向外依次是dnA核心、衣壳、皮层和囊膜.VzV皮层是核衣壳与囊膜之间一层无固定形状的蛋白层.VzV皮层由多种蛋白组成,但种类和功能仍未全部确定.本文综述了目前已发现的VzV皮层蛋白在病毒感染过程中发挥的多种作用,讨论了VzV皮层蛋白相关的蛋白-蛋白相互作用及其对病毒在SCId-Hu人鼠嵌合感染模型中组织趋向性的影响,这将有助于更好地理解VzV皮层蛋白是如何帮助病毒dnA复制、逃避宿主免疫反应和致病.福建省科技创新平台建设计划项目(批准号:2014Y2101); 厦门市科技计划项目(批准号:3502Z201410045;3502Z20131001)资

基于立即早期蛋白IE62建立水痘-带状疱疹病毒感染性滴度检测方法

目的:基于水痘-带状疱疹病毒(VZV)立即早期蛋白IE62与酶联免疫斑点检测技术(ELISpot)建立一种新型的VZV感染性滴度的快速检测方法。方法:应用生物信息学方法设计并合成VZV-IE62蛋白的多肽抗原,牛血清白蛋白偶联后免疫BALB/c小鼠,筛选和制备抗VZV-IE62单克隆抗体,应用Western blot和免疫荧光法等开展抗体性能评价,继而应用酶联免疫斑点技术(ELISpot)结合生物素-亲和素放大法建立新型的VZV感染性滴度检测方法,并与经典空斑计数法进行比较。结果:获得抗VZV-IE62的单克隆抗体1B7,应用于ELISpot方法可特异识别VZV感染后的细胞,以之为基础建立了新型的VZV感染性滴度检测方法。相比经典空斑计数法,该方法可显著缩短检测时间(从5至7 d缩短至32 h)检测结果具有较好的一致性。结论:本研究建立了一种新型的基于VZV立即早期蛋白IE62与酶联免疫斑点技术的VZV感染性滴度检测方法(VZV-IE62 ELISpot),具有潜在的转化应用前景,可为VZV的防治研究提供支持。国家自然科学基金项目(81601762)资

Preparation of monoclonal antibodies against 3D protein of EV71 based on HBc particles as expression vector

目的:预测肠道病毒71型(EV71)非结构蛋白3D的表位,以HBc蛋白为载体展示多肽,制备并鉴定抗EV71-3D的特异性单克隆抗体(mAb); 。方法:应用生物信息学方法分析预测出EV71; 3D蛋白亲水性和免疫原性指数较高的多肽片段,并运用HBc颗粒型蛋白载体展示肽段,构建多肽融合蛋白,免疫BALB/c雌鼠,通过杂交瘤技术和亲和层析; 技术制备和纯化抗EV71-3D蛋白的特异性mAb,用间接ELISA、ELISPOT、IFA和IHC对mAb的性质进行初步鉴定。结果:构建表达分别; 嵌合3D蛋白34~ 43位氨基酸残基、61~ 76位氨基酸残基、151~; 164位氨基酸残基的HBc重组蛋白,免疫并经过多轮克隆化筛选,获得抗EV71-3D单克隆抗体3E1,其亚类为IgG2a;免疫荧光试验、ELISP; OT法和免疫组织化学染色结果显示其可与EV71特异性结合。结论:成功制备可特异性识别EV71的单克隆抗体3E1,为病毒的检测及进一步研究3D蛋白; 的功能奠定了基础,同时还验证了生物信息学技术与HBc颗粒型载体展示多肽技术相结合可快速高效地制备单克隆抗体。Objective: To prepare and preliminarily identify the monoclonal; antibodies(mAbs) specifically against 3D protein of Enterovirus; 71(EV71),using bioinformatics to predict the epitopes of 3D,with HBc; protein as a carrier.Methods: Artificial screening of 3D protein epitope; sequences by bioinformatic method,inserted into the major immunodominant; region(MIR) area of Hepatitis B virus core protein(HBc),to construct the; recombinant protein.BALB/c mice were immunized with the recombinant; virus like particles(VLPs),to prepare the mAbs against 3D protein of; EV71.Affinity chromatography technology was used to purify the mAb.The; indirect ELISA,ELISPOT,immunofluorescence and immunohistochemistry; staining methods were used to identify the characteristic of the; mAb.Results: We displayed 3D(aa34-43),3D(aa61-76) and 3D(aa151-164); epitopes by constructing fusion protein using HBc VLPs as a vector,after; hybridization,one positive hybridoma cell line(3E1) was selected by; ELISA.The isotype of 3E1 was IgG2a.The results of immunofluorescence and; immunohistochemistry staining assay showed that the mAb 3E1 could; specifically recognize EV71.Conclusion: The prepared mAb 3E1 can; specifically recognizes the EV71,which laid the foundation for the; detection of virus and further study on 3D protein,and verified the; bioinformatics technology combined with HBc carrier displaying peptides; could prepare mAb quickly and efficiently.国家自然科学基金项

米非司酮对人耐药卵巢癌细胞增殖、凋亡及其对紫杉醇敏感性的影响

背景与目的:米非司酮是有效的孕酮受体拮抗剂。研究发现,米非司酮对体内外卵巢癌细胞均具有生长抑制作用,但机制尚不清楚。本研究旨在探讨米非司酮对人耐药卵巢癌细胞A2780/T增殖、凋亡及其对紫杉醇敏感性的影响,为临床应用米非司酮治疗耐药性卵巢癌提供实验依据。方法:体外培养人卵巢癌耐药细胞A2780/T,采用CCK-8法检测单用米非司酮及合用紫杉醇时对A2780/T细胞增殖的影响,分析米非司酮与紫杉醇在抑制耐药卵巢癌细胞增殖中的相互作用。采用流式细胞术分析米非司酮及米非司酮联合紫杉醇对A2780/T细胞凋亡的影响。结果:实验所选各种浓度(0.625～20μg/mL)米非司酮对A2780/T和A2780细胞均有一定程度的生长抑制作用,并呈浓度依赖性。当紫杉醇浓度为1.25或2.5μg/mL,合用米非司酮浓度为20、10、5、2.5、1.25或0.625μg/mL时,能显著抑制A2780/T细胞的增殖,并显示两种药物的协同作用(q>1.15)。当紫杉醇浓度为0.625或5μg/mL时,仅表现为两种药物的相加作用(0.85<q<1.15)。米非司酮可诱导A2780/T细胞凋亡。当米非司酮浓度为1.25、2.5和5μg/mL时,细胞凋亡率分别为(15.50±1.48)%、(26.28±0.76)%和(45.13±0.91)%。当以上3种浓度米非司酮与2.5μg/mL紫杉醇联合作用时,显示了两种药物在诱导A2780/T细胞凋亡作用上的协同效应。结论:米非司酮能够显著抑制人卵巢癌细胞A2780/T和A2780增殖,诱导A2780/T细胞凋亡,并能增强A2780/T细胞对紫杉醇的敏感性

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

Preparation and Application of Soluble Human Squamous Cell Carcinoma Antigen Expressed by Escherichia coli

旨在建立基于大肠杆菌表达系统的高效可溶性表达人鳞状上皮细胞癌抗原(SCCAg)方法,获得具有较好活性的重组SCCAg抗原并应用于建立抗原检测方法; 。基于pGEX-6P-l载体和大肠杆菌E. coli; ER2566菌株开展重组SCCAg抗原可溶性表达纯化方法研究,评价纯化抗原活性,筛选特异性单克隆抗体,初步建立并评价SCCAg抗原检测方法。结果; 显示,pGEX-6P-l载体和E coli; ER2566菌株可用于建立较高效的可溶性表达和纯化SCCAg抗原的方法,获得了具有较高纯度和活性的重组SCCAg抗原,筛选获得特异性单克隆抗体并; 初步建立了 SCCAg管式化学发光检测方法。建立了有效的基于大肠杆菌表达系统的可溶性表达和纯化SCCAg的方法。The aims of this study are to establish a method for efficient soluble; expression of human squamous cell carcinoma antigen(SCCAg ) based on; Escherichia coli expression system and obtain the recombinant SCCAg; antigen in fine activity, then apply it in the detection method; establishment of antigen. The study on the method of soluble expression; and purification of recombinant SCCAg antigen was conducted based on; pGEX-6P-l vector and E. coli ER2566 strain. The activity of the purified; antigen was evaluated by Abbott Kit and the specific monoclonal antibody; was screened by indirect ELISA. It was proved that PGEX-6P-1 vector and; E. coli strain ER2566 could be used to establish efficient soluble; expression and purification method for recombinant SCCAg antigen.; Moreover, the recombinant SCCAg antigen was proved to be in high purity; and activity. Thus,the SCCAg detection method of chemical luminous tube; was established with the specific monoclonal antibodies. In conclusion,; an effective method for the expression and purification of SCCAg, which; is based on the E. coli expression system, is established.国家高技术研究发展计划(863计划

RNA interference inhibits hepatitis B virus gene expression and replication in HepG_2-N10 cells

目的:评估以U6启动子作为启动序列(pSilencer2.0-U6)载体介导的小RNA干扰片段在培养细胞株中抑制HBV复制的效应.方法:将针对HBV基因组不同区域(S,X及C区)的核苷酸序列插入至pSilencer载体中,并将重组后的pSilencer质粒(分别为pS,pX及pC)载体转染入HepG2-N10细胞株(可稳定表达HBsAg、HBeAg及adw2亚型Dane颗粒)中.以ELISA法检测病毒抗原,以逆转录-聚合酶链反应法(reversetranscription-polymerasechainreaction,RT-PCR)检测病毒mRNA,并以荧光定量PCR法检测分泌入培养基的共价闭合环状DNA(covalentclosedcircularDNA,cccDNA).结果:质粒载体介导的RNA干扰能明显抑制培养基中HBsAg及HBeAg的表达.并且RT-PCR法检测显示病毒mRNA也被降解了,从而减少了蛋白表达及病毒逆转录复制的模板.荧光定量PCR法检测显示分泌入培养基的cccDNAs明显下降(HBVDNAlog10:pS:4.00±0.13;pC:4.08±0.10;pX:4.28±0.06;pN:5.05±0.07;HepG2-N10:4.74±0.06;HepG2:<2.70).结论:RNA干扰能抑制HBV基因表达及病毒复制,并且RNA干扰可能为HBV的治疗带来巨大的变化.AIM: To evaluate the effect of vector-based small interfering RNA promoted by U6 (pSilencer2.0-U6) on the inhibition of hepatitis B virus (HBV) replication in HepG2-N10 cells. METHODS: Several sequences targeting on dif- ferent regions of HBV genome were inserted into pSilencer vectors. The expression plasmids were then transfected into HepG2-N10 cells, a cell line which stably expressed HBsAg, HBeAg and adw2 subtype Dane Particles. Viral antigens were measured by enzyme linked immunosor- bent assay (ELISA). Viral mRNA was analyzedby reverse transcription-polymerase chain re- action (RT-PCR). The covalent closed circular DNA (cccDNA) secreted into the culture media were measured by quantitative real-time PCR. RESULTS: Vector-based RNA interference po- tently reduced HBsAg and HBeAg expression in cell culture. Furthermore, RT-PCR analysis showed that viral mRNAs were effectively de- graded, thus eliminating the messengers for pro- tein expression as well as templates for reverse transcription. Quantitative Real-time PCR analy- sis of cccDNAs revealed that vector-based RNA interference inhibited HBV replication efficiently (HBV DNA log10: pS: 4.00 ± 0.13; pC: 4.08 ± 0.10; pX: 4.28 ± 0.06; pN: 5.05 ± 0.07; HepG2-N10: 4.74 ± 0.06; HepG2: <2.70). CONCLUSION: RNA interference can inhibit HBV gene expression and virus replication

不同区段HIV-1 env基因在Bac-to-Bac昆虫细胞杆状病毒表达系统中的表达及检测

利用昆虫细胞杆状病毒表达系统 ,将从一株HIV - 1阳性克隆质粒中获得的几个HIV包膜蛋白基因片段 ,克隆入转移载体中得到重组病毒。用此重组病毒感染昆虫细胞后表达出 3种HIV包膜蛋白 ,即GP12 0 - 41P、GP41T、GP41P ,分别含有HIV - 1包膜糖蛋白GP12 0及部分GP41,删除了N端 12个疏水氨基酸的GP41和仅有主要表位约 2 40个氨基酸的GP41。收获后分别以Western -blotting和EIA检测 ,有较好的免疫学活性 ,其中GP41T的活性最强。该实验为HIV包膜蛋白的结构研究提供了依据 ,加以改进后可能有免疫检测的价值

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