表达绿色荧光蛋白的重组耻垢分枝杆菌感染THP-1巨噬细胞模型的建立

目的 建立表达绿色荧光蛋白(GFP)的耻垢分枝杆菌感染THP-1巨噬细胞模型,对耻垢分枝杆菌进行快速定位和直观检测,为探究结核发病机制以及研制新型结核疫苗提供一种示踪工具。方法 以pEGFP-N1质粒为模板采用PCR扩增出增强型绿色荧光蛋白(EGFP)基因序列,获得EGFP的编码基因,将扩增产物克隆到载体pALACE上,建立重组质粒pALACE-EGFP。利用电穿孔技术将pALACE-EGFP转化到耻垢分枝杆菌中,通过潮霉素抗性筛选重组耻垢分枝杆菌克隆,扩大培养后涂片观察,并进行荧光显微镜镜检。再用重组耻垢分枝杆菌感染THP-1巨噬细胞,观察是否有GFP表达。结果 正确构建重组质粒pALACE-EGFP;荧光显微镜下观察重组耻垢分枝杆菌,证实EGFP在重组耻垢分枝杆菌中表达,并且THP-1巨噬细胞被感染后发出绿色荧光。结论 表达EGFP蛋白的重组耻垢分枝杆菌的成功建立,为结核分枝杆菌的感染和致病机制研究奠定基础。     

重组耻垢分枝杆菌对小鼠结核病免疫治疗效果的评价

目的 评估携带编码人天然颗粒溶素(GLS)和小鼠IL-12基因质粒(pZM03)的重组耻垢分枝杆菌对结核分枝杆菌感染的治疗效果,及其与临床抗痨药物疗效的比较.方法 结核分枝杆菌H37Rv感染Balb/c小鼠4周后分别用生理盐水、GLS/IL-12重组耻垢分枝杆菌(重组M.S)、INH PZA和重组M.S INH PZA治疗,于第1次治疗后3个月,处死小鼠,检测器官荷菌量、脾淋巴细胞IFN-γ和INF-α的分泌水平、血清IL-12和IFN-γ分泌水平、肺泡灌洗液SIgA的水平和肺、脾组织中GLS表达,同时观察小鼠肺、脾组织病理改变情况.结果 重组M.s、INH PZA和重组M.s INH PZA治疗组肺、脾组织荷菌量(log CFU/g)比生理盐水组显著降低.重组M.s组经PPD刺激后IFN-γ和TNF-α分泌水平明显高于其它组.重组M.s组血清IL-12和IFN-γ水平明显高于其它组.各组产生黏膜特异性SIgA明显高于未经感染组.用免疫组化检测到小鼠肺及脾组织中GKS的表达.生理盐水组肺组织病理改变以渗出为主,病变广泛,正常肺泡结构被破坏;其余组肺组织病变轻且局限.结论 重组M.s对小鼠结核病有一定免疫治疗作用,通过增强宿主Th1型免疫应答和GLS的抗菌活性有关;重组M.S对临床常用的抗结核药有协同作用,为结核病的综合治疗打下实验基础.     

结核分枝杆菌Rv0901基因重组耻垢分枝杆菌的构建及其细胞作用

目的对结核分枝杆菌Rv0901基因的功能进行研究。方法以PCR扩增Rv0901基因编码序列,定向克隆人穿梭表达质粒pMV261获得重组穿梭表达质粒,将重组质粒电穿孔进入耻垢分枝杆菌,构建重组Rv0901基因的耻垢分枝杆菌,对重组耻垢分枝杆菌进行诱导表达,用SDS-PAGE检测表达结果。比较耻垢分枝杆菌和重组耻垢分枝杆菌对THP-1细胞的不同作用。结果成功构建重组穿梭表达质粒及重组Rv0901基因的耻垢分枝杆菌,重组菌诱导THP-1细胞的凋亡率高于耻垢分枝杆菌,其感染THP-1细胞后细胞的存活率低于耻垢分枝杆菌的感染,重组菌感染THP-1细胞后细胞培养液中NO(一氧化氮)的产生高于耻垢分枝杆菌的感染。结论Rv0901基因可能与结核毒力存在一定关系。     

构建及鉴定TACO特异性10-23DRz表达载体重组耻垢分枝杆菌

目的 构建一种可表达特异性10-23脱氧核酶(deoxyribozyme,DZ)的巨噬细胞靶向性细菌载体疫苗,并鉴定其在巨噬细胞表达10-23DZ的能力以及抑制靶基因表达的效果.方法 采用亚克隆方法将分枝杆菌复制子oriM克隆入10-23DRz真核表达载体pSDE01中,获取可在分枝杆菌和真核细胞中穿梭的重组质粒pSDE02.在前期研究基础上选择一有效的针对巨噬细胞taco基因的10-23DZ--DZ1,根据DZ1序列设计并制备DZ1的双链表达序列,插入pSDE02中获取重组质粒pDZM01.将pDZM01经电穿孔方法转化入耻垢分枝杆菌Ms1-2c株构建重组耻垢分枝杆菌rMS-DZ1.分别采用Ziehl-Heelson染色法和直接荧光观测法鉴定重组耻垢分枝杆菌的巨噬细胞靶向性.用rMS-DZ1感染巨噬细胞株RAW264.7,分别于感染后的24 h和48 h采用斑点杂交方法检测rMS-DZ1在巨噬细胞中表达DZ1的情况,再分别采取RT-PCR和免疫印迹方法检测巨噬细胞TACO表达的差异.结果 限制性内切酶酶切、菌落PCR及测序结果显示pSDE02、pDZM01和rMS-DZ1构建成功.rMS-DZ1感染RAW264.7细胞后24 h和48 h,经点杂交方法均检测到了DZ1的表达.半定量RT-PCR和免疫印迹检测结果显示,与未感染组比较,rMS-DZ1感染后24 h和48 h时巨噬细胞TACOmRNA分别下降了67.90%和57.14%,TACO蛋白水平分别下降了53.85%和68.92%.同时,表达对照寡脱氧核糖核苷酸DZ1U的重组耻垢分枝杆菌rMS-DZ1U感染RAW264.7细胞后,其TACOmRNA和TACO蛋白水平与未感染组比较均无明显差异.结论 本研究成功构建了一种可表达特异性10-23DZ的巨噬细胞靶向性重组耻垢分枝杆菌的载体,可在巨噬细胞内表达并抑制相应靶基因的表达,可作为研制治疗用疫苗的基础,这也是国内外首次报道可表达10-23DZ的细菌性载体.

Abstract：

Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.     

GLS和IL-12偶联重组耻垢分枝杆菌免疫效果的观察

目的：研究携带编码人天然颗粒溶素（GLS）和小鼠IL-12基因质粒（pZM03）偶联重组耻垢分枝杆菌（ATCC607）经鼻黏膜免疫小鼠后,小鼠体内的免疫状况.方法：BALB/c小鼠36只,随机分为生理盐水、pZM03、ATCC607、卡介苗（BCG）、重组ATCC607（即携带pZM03的ATCC607）、灭活重组ATCC607组;采用滴鼻法免疫小鼠,BCG组0、14天各1次,其它组0、4、14天各1次,第0天免疫后4周处死小鼠,用ELISA检测血清中IFN-γ、IL-12、IgG2a、lL-4的分泌水平和淋巴细胞PPD诱导的IFN-γ分泌水平以及肺泡灌洗液特异性SIgA的水平,用MTS法检测免疫小鼠脾淋巴细胞增殖情况.结果：重组ATCC607血清中IFN-γ、IL-12水平与BCG组无明显差异但明显高于其他组（P＜0.05）,IgG2a水平重组ATCC607组高于BCG组和其他组（P＜0.05）,各组间IL-4水平差异无统计学意义.重组ATCC607、BCG、灭活重组ATCC607及ATCC607组用PPD均可诱导小鼠脾淋巴细胞增殖,各组间差异无显著意义,但与生理盐水和pZM03组间差异有显著意义（P＜0.05）.重组ATCC607组PPD诱导淋巴细胞IFN-γ明显高于其它组（P＜0.05）,与BCG组比较无显著差异.重组ATCC607等含菌组经鼻黏膜免疫可产生黏膜特异性SIgA.结论：重组ATCC607经鼻黏膜免疫小鼠后,机体特异性免疫特别是细胞免疫和黏膜免疫增强,为重组ATCC607治疗结核病的研究奠定了基础.     

耻垢分枝杆菌作为免疫调节剂的研究

目的 探讨耻垢分枝杆菌制剂对不同免疫状态动物的免疫调节作用.方法 注射环磷酰胺建立免疫功能低下的动物模型,以结核分枝杆菌感染（早期）或猪血清致敏建立免疫功能亢进的动物模型,然后将耻垢分枝杆菌制剂分别注射于正常、免疫功能低下和免疫功能亢进的动物,研究耻垢分枝杆菌制剂对不同模型动物T淋巴细胞增殖反应、迟发型超敏反应或速发型超敏反应的影响.结果 耻垢分枝杆菌制剂可增强正常动物的T淋巴细胞增殖反应和迟发型超敏反应,促进免疫功能低下小鼠T淋巴细胞增殖功能的恢复,抑制免疫功能亢进豚鼠的迟发型超敏反应和猪血清致敏小鼠的速发型超敏反应.结论 耻垢分枝杆菌制剂具有双向免疫调节作用.     

重组耻垢分枝杆菌Ag85B-ESAT6-rMs对小鼠巨噬细胞功能的影响

目的:探讨重组耻垢分枝杆菌Ag85B-ESAT6-rMs对小鼠巨噬细胞功能的影响。方法:将耻垢分枝杆菌(Ms)与重组耻垢分枝杆菌Ag85B-ESAT6-rMs分别感染小鼠巨噬细胞系RAW264.7,通过抗酸染色和细胞内细菌菌落形成单位计数评价Ms和Ag85B-ESAT6-rMs被巨噬细胞吞噬和消化能力;采用流式细胞术检测巨噬细胞凋亡情况;分别采用实时定量PCR技术和ELISA检测细胞因子TNF-α的表达和分泌水平。结果:小鼠巨噬细胞实验证实,Ag85B-ESAT6-rMs比Ms具有更强的感染和促进巨噬细胞的吞噬能力,Ag85B-ESAT6-rMs可增加巨噬细胞表达和分泌TNF-α的水平,并促进受感染巨噬细胞的凋亡。结论:Ag85B-ESAT6-rMs可以促进巨噬细胞的吞噬消化功能,将有助于增强巨噬细胞对Ms抗原的递呈和机体抗Ms感染的特异性免疫。     

重组耻垢分枝杆菌制剂预防幽门螺杆菌感染的作用机理的初步探讨

目的探讨重组耻垢分枝杆菌实验制剂（recombinant Mycobacterium smegmatis，rMs）预防幽门螺杆菌（Helicobacter pylori，Hp）感染的作用机理。方法实验制剂免疫BLAB／c小鼠4周，用Hp攻击后4周，取血清、胃、脾组织，RT-PCR检测小鼠胃黏膜和脾组织的TH1型细胞因子（IFN-γ、IL-2、IL-12）与TH2型细胞因子（IL-4、IL-6、IL-10）；用ELISA检测针对唧的特异性血清IgG、IgG1、IgG2a和IgA水平；用免疫组化方法检测胃黏膜固有层IgA表达；用MTT检测脾淋巴细胞增殖。结果免疫后BALB／c小鼠特异性抗体显示rMs制剂诱导Hp特异性血清IgG、IgA水平都升高，以IgG2a占优势。用Hp抗原刺激后各免疫组小鼠脾淋巴细胞均有明显增殖。免疫组化显示各免疫组小鼠胃黏膜固有层IgA阳性标记腺体数明显高于对照组。RT-PCR证实，跏攻击之前，各免疫组胃和脾组织已出现了IFN-γ、IL-12、IL-2表达而无IL-4、IL-6、IL-10表达。PBS对照组和单纯耻垢分枝杆菌（Ms）组胃黏膜和脾组织各细胞因子均无表达；Hp攻击后，PBS和单纯Ms组胃组织出现以TH1细胞IFN-γ为主的增生，IFN-γ水平显著高于rMs组（P〈0．05）；而3个rMs制剂组脾脏则出现以TH2细胞（IL-4）为主的增生，IL-4水平显著高于对照组（P〈0．05）。提示该制剂的作用机制是诱导TH1，TH2平衡型免疫应答。结论成功地建立了BALB／c小鼠的Hp感染模型，对rMs制荆的免疫保护机理研究结果显示，rMs能产生以TH1细胞和TH2细胞协同作用的保护性免疫应答。     

GLS/IL-12重组耻垢分枝杆菌的构建及其在小鼠体内的表达

目的 构建携带编码人天然颗粒溶素(GLS)和小鼠IL-12基因质粒(pZM03)的重组耻垢分枝杆菌,并经鼻黏膜免疫观察其在小鼠体内的表达.方法 将人GLS和小鼠IL-12基因及分枝杆菌复制子OriM同时克隆进多启动子真核共表达载体pBudCE4.1中,得到穿梭质粒pZM03,以pZM03电转化耻垢分枝杆菌,再将该重组耻垢分枝杆菌滴鼻免疫Balb/c小鼠3次,第1次免疫后4周,处死小鼠,用免疫组化检测肺、脾组织中GLS表达,用ELISA检测血清IL-12和肺泡灌洗液特异性SIgA的水平.结果 得到可表达GLS/IL-12重组耻垢分枝杆菌;在Balb/c小鼠肺、脾组织中均检测到重组耻垢分枝杆菌的分布,以及GLS的表达,并有血清IL-12水平升高和黏膜特异性SIgA的产生.结论 成功构建GLS和IL-12修饰的重组耻垢分枝杆菌,该重组菌经鼻黏膜免疫小鼠后,可引起呼吸道黏膜特异性抗菌免疫,以及GLS和IL-12的体内表达,为新型疫苗的研究奠定了基础.     

结核杆菌CFP-10/ESAT-6融合基因在耻垢分枝杆菌中的表达及其抗原性的初步研究

目的构建表达结核分枝杆菌CFP-10／ESAT-6融合蛋白的重组耻垢分枝杆菌,并观察其对巨噬细胞的作用。方法采用SOE法构建CFP-10／ESAT-6融合基因,克隆人大肠杆菌-分枝杆菌穿梭载体pMV261中,将重组质粒电转化耻垢分枝杆菌,SDS-PAGE检测CFP-10／ESAT-6融合蛋白在重组耻垢分枝杆菌中的表达。以重组耻垢分枝杆菌感染小鼠巨噬细胞ANA-1,半定量RT-PCR检测ANA-1细胞一氧化氮合成酶的表达水平。结果经DNA测序证实CFP-10／ESAT-6融合基因序列正确。重组耻垢分枝杆菌经热诱导后可以表达相对分子质量(Mr)约为18×103的融合蛋白,与预期值一致。重组耻垢分枝杆菌能够诱导小鼠巨噬细胞表达一氧化氮合成酶。结论表达CFP-10／ESAT-6融合蛋白的重组耻垢分枝杆菌构建成功,该重组耻垢分枝杆菌能够活化巨噬细胞,具有抗原性,为研制结核疫苗提供一定参考。     

Flag与GFP双标记人微粒体甘油三酯转移蛋白重组腺病毒载体的构建与表达

目的利用重组腺病毒（adenovirus，ad）技术Adeasy系统构建带绿色荧光蛋白（Green fluorescence protein，GFP）和Flag双标记的人类微粒体甘油三酯转移蛋白（human microsomal triglyceride transfer protein，hMTP）基因重组腺病毒载体，并包装成高滴度的腺病毒。方法自人HepG2细胞获得RNA，以RT-PCR和Nested PCR扩增hMTP-flag基因。PCR产物经测序证实后插入穿梭质粒GFP-Track。经PmeⅠ酶切线性化后，共转化到含腺病毒骨架质粒Adeasy的感受态细菌BJ5183。筛取阳性克隆，经PacⅠ酶切线性化后转染293A细胞，产生包装病毒颗粒，并传代扩增培养。结果经测序证实得到hMTP-flag-GFP基因，限制性内切酶证实同源重组腺病毒包含目的基因。荧光显微镜下可见GFP的高效表达，Western-blot检测可见97KD目的条带（hMTP分子质量为97KD）。结论成功构建含hMTP-flag-GFP基因的双标记重组腺病毒载体，并进行蛋白质表达，为进一步研究脂蛋白的功能打好了坚实基础。     

hsa—miR-30e的克隆及其慢病毒表达载体的构建

目的克隆hsa—miR-30e并构建其慢病毒表达载体。方法PCR扩增hsa—miR-30e的前体序列,克隆于慢病毒载体p113．7,转染包装细胞293FT细胞,收获病毒颗粒,荧光定量PCR检测hsa—miR-30e的表达。结果经双酶切及测序鉴定,hsa—miR-30e的慢病毒表达载体构建成功,倒置荧光显微镜下观察转染后293FT细胞发出绿色荧光。结论成功构建hsa—miR-30e的慢病毒表达载体,为进一步研究hsa-miR-30e的生物学功能奠定了基础。     

Recombinant Mycobacterium bovis BCG Expressing the Chimeric Protein of Antigen 85B and ESAT-6 Enhances the Th1 Cell-Mediated Response

The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant Mycobacterium bovis BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-A_N-E-A_C). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-γ) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4⁺ and CD8⁺ T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-A_N-E-A_C significantly increased the level of production of the major Th1 cytokines IFN-γ and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-A_N-E-A_C was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-A_N-E-A_C strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against M. tuberculosis.Mycobacterium bovis bacillus Calmette Guérin (BCG) is the only vaccine against tuberculosis (TB) currently available and exhibits various levels of efficacy for the prevention of pulmonary TB (range, 0 to 80%) in different trials (9). BCG has a protective effect in children, particularly against tuberculous meningitis; however, it does not satisfactorily prevent the development of pulmonary TB in adults and fails to protect individuals against reinfection (1). Given the rate of mortality from TB worldwide, with more 8 million new cases and 2 million deaths occurring annually (2), newer strategies need to be implemented to improve BCG or vaccines more effective than BCG urgently need to be developed.One approach that might be used to increase the efficacy of BCG could be to construct a recombinant BCG (rBCG) which either overexpresses immunogenic antigens or modulates the ensuing immune response (8). rBCG vaccines are attractive because of the widespread experience with their use, the known immunogenicity associated with protection against the worst forms of the disease in children, and the safety profiles of standard BCG strains (13). Two rBCG vaccines have been entered into clinical trials. This includes rBCG30, which expresses the antigen 85B (Ag85B) protein, and ΔureC hly-positive rBCG, which expresses listeriolysin and which is urease deficient (12, 15). It is hoped that these vaccines will provide a strong and perhaps longer-lasting immune response than that achieved with the conventional BCG vaccine.The most effective defined-antigen TB vaccines will likely require the induction of both cell-mediated and humoral immune responses. Ag85B and the 6-kDa early secreted antigen target (ESAT-6) have been identified as two of the most promising vaccine candidates which are strongly recognized by T lymphocytes (3, 19). In a previous study, we relied on the T-cell epitopes of Ag85B and ESAT-6 to design a chimeric protein by inserting ESAT-6 into Ag85B from amino acids 167 to 182 and demonstrated that this recombination of Ag85B and ESAT-6 could improve the immunogenicity and enhance the T-helper type 1 (Th1) cell-mediated immune response (27). This finding prompted us to explore further the efficacy of rBCG overexpressing this chimeric protein. In this study, we constructed rBCG expressing chimeric protein Ag85B_N-ESAT-6-Ag85B_C (rBCG-A_N-E-A_C) and further compared the immune response to that protein with that to rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) and BCG.     

TIMP-3N端结构域重组腺病毒载体的构建与鉴定

目的 构建N-TIMP3重组腺病毒载体,为深入研究组织金属蛋白酶抑制因子-3(tissue inhibitor of metalloproteinases-3,TIMP-3)N端结构域对胰腺癌的作用奠定基础.方法 PCR扩增编码人TIMP3信号肽及其N端结构域的DNA序列,定向克隆至空载穿梭质粒pShuttle-CMV,构建重组穿梭质粒pShuttle-N-TIMP3,并测序鉴定.将重组pShuttle-N-TIMP3转化BJ5183感受态细菌,与其内的腺病毒骨架质粒pAdEasy-1进行同源重组,构建重组腺病毒载体pAd-N-TIMP3,并经酶切与PCR鉴定.将线性化的重组载体转染QBI-293A细胞包装病毒,TCID50法检测病毒滴度,用Western 印迹检测目的 蛋白的表达.结果 重组腺病毒载体pAd-N-TIMP3经鉴定正确,病毒滴度为5×108 PFU/ml,并检测到目的 蛋白的表达.结论 成功构建了重组腺病毒载体pAd-N-TIMP3.     

t-PA基因新型真核表达载体的构建及鉴定

目的构建人组织型纤溶酶原激活因子(tPA)基因的新型真核表达载体,为血栓性疾病的基因治疗奠定基础。方法将tPA基因克隆到真核表达载体pcDNA3.1MycHisB()中,进行酶切鉴定及DNA测序分析,并将携带有tPA基因的该真核表达载体,通过脂质体介导,转染鼠血管平滑肌细胞,底物发色法测定转基因血管平滑肌细胞tPA活性。结果经双酶切鉴定和测序证实已将tPA基因DNA片段正确插入到真核表达载体中,并能在真核细胞中表达、分泌有活性的tPA。结论成功构建pcDNA3.1MycHisB()/tPA基因新型真核表达载体。     

Construction and Immunogenicity of Recombinant Adenovirus Vaccines Expressing the HMW1, HMW2, or Hia Adhesion Protein of Nontypeable Haemophilus influenzae

The objective of the present study was to construct and assess the immunogenicity of recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia protein of nontypeable Haemophilus influenzae (NTHi). These proteins are critical adhesins and potential protective antigens expressed by NTHi. Segments of the hmw1A and hmw2A structural genes that encode the distal one-half of mature HMW1 or HMW2 were cloned into the T7 expression vector pGEMEX-2. These constructs encoded stable HMW1 or HMW2 recombinant fusion protein that expresses B-cell epitopes common to most NTHi strains. A segment of the hia gene that encodes the surface-exposed portion of mature Hia was also cloned into pGEMEX-2. The resulting T7 gene 10 translational fusions were excised from the parent plasmids and cloned into the shuttle plasmid pDC316. Cotransfection of HEK 293 cells with the pDC316 derivatives and pBHGloxΔE1,3Cre resulted in the production of viral plaques from which recombinant adenoviruses expressing fusion proteins were recovered. Chinchillas immunized intraperitoneally with a single 10⁸-PFU dose of either the HMW2 or Hia adenoviral construct developed high anti-HMW2 or anti-Hia serum antibody titers within 4 weeks of immunization. Chinchillas immunized intranasally with a single 10⁷- to 10⁹-PFU dose of the Hia adenoviral construct also developed high anti-Hia serum antibody titers within 8 weeks of immunization. Recombinant adenoviruses represent a promising system to induce mucosal and systemic immunity and protection against mucosal diseases such as otitis media. Recombinant adenoviruses expressing recombinant HMW1, HMW2, or Hia protein will be important new tools in NTHi vaccine development efforts.Otitis media remains a significant health problem for children in this country and elsewhere in the world (15, 16). Most children in the United States have had at least one episode of otitis by the third birthday, and one-third have had three or more episodes (51). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (50). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for prevention of otitis media in young children (26).Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of cases of acute otitis media (20, 53). Streptococcus pneumoniae has been the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (20, 53). Nontypeable Haemophilus influenzae (NTHi) is the second most common bacterium recovered and accounts for 20% to 30% of cases of acute otitis media and a larger percentage of cases of chronic and recurrent disease (37). Interestingly, since introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccination schedule, nontypeable Haemophilus influenzae has become an even more common cause of acute and recurrent middle ear disease, often surpassing Streptococcus pneumoniae in frequency of recovery from middle ear fluid specimens (12, 18).Many different antigens have been suggested as possible nontypeable Haemophilus influenzae vaccine candidates (1, 5, 23, 43, 44, 63). In our early work, we demonstrated that development of bactericidal antibody in the sera of children who had recovered from acute NTHi otitis media was associated with the appearance of serum antibodies directed against highly immunogenic high-molecular-weight (HMW) proteins (7). This work led subsequently to the identification and characterization of the HMW1 and HMW2 (HMW1/HMW2) family of proteins (8). The HMW1/HMW2 proteins have subsequently been shown to be major adhesins of nontypeable Haemophilus influenzae (57) as well as targets of opsonophagocytic (65, 66) and protective (6) antibodies. The HMW1/HMW2-like proteins are expressed by approximately 75% of NTHi strains (8, 58). The 25% of NTHi strains that do not express HMW1/HMW2-like proteins also express immunogenic high-molecular-weight proteins that are recognized by human convalescent-phase serum antibodies (11). Almost all the HMW1/HMW2-negative strains have subsequently been shown to express a second distinct class of adhesins known as Hia proteins (11). The Hia proteins are members of a large family of bacterial proteins known as autotransporters that are found in many Gram-negative bacteria (28, 69). The Hia proteins have also recently been shown to serve as targets for opsonophagocytic antibodies (64). Nearly all NTHi strains that lack HMW1/HMW2 proteins contain a hia gene and express a Hia protein, and conversely, strains that express HMW1/HMW2 proteins lack a hia gene (11, 58).Several groups have begun exploring mucosal and, in particular, nasopharyngeal immunization strategies to stimulate a protective immune response in the upper respiratory tract and middle ear (19, 24), and results to date have been encouraging (4, 29, 32, 47). Intranasal immunization has a number of potential advantages over traditional parenteral immunization approaches for prevention of otitis media. The presence of abundant microvilli in the nasal cavity greatly increases the available surface area of this anatomical site, thereby generating a large absorptive surface (34). Immunization via the nasal cavity also allows direct delivery of immunogens of interest to inductive/effector sites (i.e., nasal mucosa-associated lymphoid tissue [NALT] and Waldeyer''s ring), which can result in both a systemic and a mucosal response (14, 33, 62). Furthermore, the nasal cavity is readily accessible and allows for noninvasive delivery of antigens or vaccines, thus eliminating the need for trained staff or the use of sterile needles and syringes. The option of treating or immunizing against human disease with such a noninvasive technique would almost certainly result in increased patient compliance if intranasal vaccines advance to the clinic.A number of mucosal vaccination strategies continue to be actively explored (41, 49). Adenoviruses have been identified as promising live recombinant vaccine vectors based upon their ability to induce high levels of heterologous gene expression (13, 25, 55, 60) and to stimulate mucosal immunity in the upper respiratory tract, their natural site of replication. E1-deletion-containing replication-defective adenoviral recombinants based on human serotype 5 (Adhu5) have been tested as vaccine candidates for prevention of a number of infectious diseases (21, 60). Studies of adenoviral vaccines based upon the H5N1 hemagglutinin protein (30), surface proteins of Streptococcus pneumoniae (3), the rabies virus glycoprotein (68), the circumsporozoite protein of Plasmodium falciparum (54), and the E6 and E7 oncoproteins of human papillomavirus type 16 (HPV-16) (27) have demonstrated that E1-deletion-containing vaccines induce excellent B-cell and CD8⁺-T-cell responses in experimental animals, even if given at moderate doses.Recombinant adenovirus (rAd) vectors have yet to be investigated as potential vaccine candidates for prevention of otitis media. However, they are particularly attractive candidates for the reasons noted above. The objective of the present study was to construct E1-deletion-containing replication-defective recombinant adenovirus vectors expressing the HMW1, HMW2, or Hia adhesion protein of nontypeable Haemophilus influenzae and to assess their immunogenicity in the chinchilla experimental model.     

鼠内皮抑素基因腺病毒载体的构建及对血管内皮细胞的生长抑制

构建含鼠内皮抑素基因的腺病毒载体（pAd／mEnd），并观察内皮抑素蛋白对人脐静脉血管内皮细胞（HUVEC）的生长抑制、细胞周期和凋亡的影响。结果发现pAd／mEnd感染BEL7402细胞后，能有效表达内皮抑素蛋白，显著抑制HUVEC增殖，阻滞HUVEC细胞S期，凋亡指数显著增加。     

IFITM3真核表达载体的构建和细胞内定位研究

目的构建人干扰素诱导的跨膜蛋白3(IFITM3)的真核表达载体并观察其表达产物的细胞内定位,以研究IFITM3的蛋白功能及作用机制。方法提取人外周血淋巴细胞mRNA,行RT-PCR扩增IFITM3编码序列并经琼脂糖凝胶电泳鉴定片段大小,PCR产物及真核表达载体pEGFP-C2经酶切、连接、转化入Top10菌株进行筛选和扩增,重组的pEGFP-C2-IFITM3载体用酶切及DNA测序鉴定其插入序列的正确性,进而转染该重组真核表达载体进入sy5y细胞,倒置荧光共聚焦显微镜观察融合蛋白在细胞内定位。结果琼脂糖凝胶电泳显示经RT-PCR的扩增产物与IFITM3编码区的实际长度相吻合,重组入绿色荧光载体pEGFP-C2的pEGFP-C2-IFITM3经酶切和测序显示实际连入的DNA片段大小、序列及读码框均正确。经真核表达后,重组蛋白定位于细胞膜和在细胞内存在颗粒样聚集。结论成功构建IFITM3的真核表达载体,该载体表达的IFITM3融合蛋白在细胞内定位清晰,为后续的IFITM3功能研究奠定了基础。     

GFP／HBVX融合蛋白重组载体的构建及稳定表达细胞系的建立

目的构建绿色荧光蛋白（GFP）与人乙型肝炎病毒（HBV）X基因的重组表达载体，建立稳定表达HBVX蛋白（HBx）与GFP融合蛋白的HepG2细胞系，以进一步研究HBx的生物学功能及其在肝癌发生中的作用。方法应用PCR法从adr亚型HBV质粒pHBVDNA中扩增HBVX基因片段，PCR产物经HindⅢ和KpnⅠ双酶切后定向插入绿色荧光蛋白真核表达载体pEGFP-C1的相应酶切位点，转化宿主菌DH5α，采用上述双酶切及DNA测序鉴定重组质粒pGFP-HBx；采用脂质体转染法将pEGFP-C1质粒、pGFP-HBx重组质粒DNA转染人肝母细胞瘤细胞系HepG2细胞，G418选择抗性细胞克隆，荧光显微镜下观察GFP表达，挑取表达GFP的抗性克隆扩大培养、传代。采用RT-PCR检测转染细胞HBVX基因的表达。结果经酶切及测序鉴定成功构建了GFP-HBVX重组表达载体pGFP-HBx；将pEGFP-C1、pGFP-HBx重组质粒转染HepG2．，经G418筛选15d获得抗性细胞克隆。将带绿色荧光的抗性克隆细胞扩大培养并经传代70次，细胞仍表达强的荧光蛋白。RT-PCR检测表明转染pGFP-HBx重组体的HepG2／GFP-HBx细胞有HBVX转录、表达。结论成功构建了GFP．HBVX真核重组表达载体pGrP-HBx；获得了稳定表达GFP-HBx融合蛋白的HepG2细胞系，这为进一步研究FIBx的生物学功能以及HBx在肝癌发生中的作用与机制打下了基础。     

Protection against Mycobacterium avium by DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.