多型别HCV-E1复合表位抗原研究

目的 设计多型别HCV-E1表位复合免疫原,通过免疫小鼠,探讨其在丙型肝炎治疗性疫苗与诊断试剂研究中的应用.方法 分析比较HCV-E1包膜糖蛋白B细胞表位序列,选取几个代表性基因型的中和性优势抗原表位,再结合泛DR辅助性T细胞表位(PADRE),构建含有不同HCV型别E1表位抗原基因和通用T辅助细胞表位基因的原核表达质粒.结果 成功构建了含有HCV 1a、1b、2a、3a、4a和6a等基因型E1中和性表位以及通用T辅助细胞表位的质粒pBVIL1/E1s-PADRE,该质粒转化大肠杆菌后获得的工程菌,通过诱导培养可以高效表达重组多型别HCV-E1表位复合抗原,所获得的多型别HCV-E1表位复合免疫原可在BALB/c小鼠体内诱发强烈的体液免疫反应,ELISA检测抗体水平可达到1:12 800.结论 新构建的HCV-E1表位复合免疫原具有很好的免疫原性,为进一步研究HCV疫苗奠定了基础.     

抗BCG Hsp65单克隆抗体的鉴定及应用研究

目的:对制备的5株抗BCG Hsp65单克隆抗体(Hsp65 mAb)做进一步鉴定和应用.方法:采用ELISA方法对mAb的效价、亚类和结合抗原表位特性进行鉴定,应用mAb对Hsp65融合蛋白进行了Western blot和免疫荧光检测.结果:5株mAb的类型均为IgG,其中ⅠC1和ⅡC3株mAb为IgG1亚类,ⅠA5、ⅠC3和ⅠD6株mAb为IgG2a亚类. 抗原表位竞争结合分析显示,在配对的不同亚类mAb中,存在着结合Hsp65表面不同抗原表位的mAb.通过Western blot检测证实,Hsp65 mAb对Hsp65-MUC1检测结果,与MUC1 mAb的一致.细胞免疫荧光检测显示,制备的 mAb能够识别通过蛋白装载进入树突状细胞内的Hsp65-PSA.结论:获得的Hsp65 mAb可做为检测工具,用于研究BCG Hsp65及其融合蛋白的功能和表达.     

共刺激分子4-1BBL基因免疫对HBsAg核酸疫苗诱导小鼠特异性免疫应答的影响

目的 研究共刺激分子4-1BBL基因免疫对HBsAg核酸疫苗诱导小鼠特异性体液和细胞免疫应答的影响.方法 将HBV表面抗原核酸疫苗pcDS2单独或联合共刺激分子4-1BBL质粒肌肉注射免疫C57BL/6小鼠;ELISA法检测小鼠血清抗-HBs IgG及亚型IgG1和IgG2a;迟发型超敏反应(DTH)反应检测体内细胞反应;流式细胞仪检测CD4+ T淋巴细胞分泌IL-4和IFN-γ及CD8+T淋巴细胞分泌IFN-γ水平;流式细胞仪检测小鼠脾细胞HBsAg特异性体外细胞毒性T淋巴细胞杀伤作用(CTL).结果 与单纯免疫核酸疫苗pcDS2组比较,pcDS2和4-1BBL联合免疫组小鼠的抗-HBs水平显著提高,抗-HBs IgG亚类以IgG2a占优;免疫小鼠经HBsAg脚掌皮下刺激后,联合免疫组小鼠脚掌的厚度显著高于pcDS2组;联合免疫组CD4+T淋巴细胞的IL-4和IFN-γ表达水平及CD8+T淋巴细胞的IFN-γ表达水平显著升高;DNA疫苗免疫的各组小鼠,HBsAg特异性体外CIL杀伤作用高于对照组,其中联合免疫组小鼠的体外CTL杀伤作用最强.结论共刺激分子4-1BBL不仅能增强HBV DNA疫苗诱导特异性体液免疫应答,还能增强特异性型细胞免疫反应,尤其增强体内CIL的杀伤活性.     

乙型肝炎病毒治疗性多表位基因疫苗在小鼠中的免疫应答研究

目的:分析乙型肝炎病毒(HBV)重组多表位抗原基因在大肠杆菌中非融合表达的蛋白B-BPT的免疫原性,以期为HBV预防/治疗性疫苗的研制奠定实验基础。方法:构建重组质粒pBAD/BPT,并在大肠杆菌中诱导表达。以纯化的蛋白B-BPT免疫BALB/c小鼠,用乳酸脱氢酶(LDH)释放法检测该融合蛋白诱导的小鼠细胞毒T淋巴细胞(CTL)应答。用流式细胞术(FCM)检测小鼠CD4+、CD8+T淋巴细胞亚群的比例,用MTT比色法检测淋巴细胞的增殖效应。结果:B-BPT蛋白免疫BALB/c小鼠后,可诱导特异性CTL应答(P<0.05,与空白对照组比较);B-BPT蛋白免疫后,可显著刺激小鼠脾淋巴细胞增值(P<0.05,与空白对照组比较)。结论:该非融合表达的蛋白B-BPT可有效地诱导小鼠特异性CTL免疫应答,为进一步研制乙肝预防治疗性疫苗奠定了基础。     

应用ELISPOT方法筛选确定尼帕病毒融合糖蛋白和附着糖蛋白的H-2~d限制性T细胞表位

目的在小鼠中筛选和确定尼帕病毒(Nipah virus, NiV)融合糖蛋白(fusion glycoprotein, F)和附着糖蛋白(attachment glycoprotein, G)特异性的H-2d限制性T细胞表位。方法本研究基于NiV F和G蛋白全序列设计合成多肽库(单肽长为15个氨基酸, 前后肽重复10个氨基酸), 使用表达NiV F和G蛋白的DNA疫苗肌肉注射免疫BALB/c小鼠, 通过矩阵设计将F和G抗原全序列肽库分别混合成肽池, 取免疫后小鼠脾细胞进行IFN-γ ELISPOT检测, 确定肽库中特异反应的优势表位肽。结果 F肽库筛选到12条特异性优势肽, 第56条多肽产生的反应最强;G肽库筛选到4条特异性优势肽, 第72条多肽产生的反应最强。结论本研究使用IFN-γ ELISPOT方法筛选和确定了12条NiV F抗原特异性和4条NiV G抗原特异性H-2d限制性优势T细胞表位, 为NiV免疫学与疫苗研究提供了参考。     

B7-H1蛋白疫苗对HBV转基因小鼠HBsAg免疫效果影响的初步研究

目的:应用HBV转基因小鼠动物模型,研究B7-H1对HBsAg免疫效果的影响.方法:用重组人B7-H1与血源性HBsAg联合免疫HBV转基因小鼠,采用ELISA方法观察对转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的影响,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γ T细胞数量的影响,同时检测对小鼠淋巴细胞增殖及血清HBsAb水平的影响.结果:HBsAg组及HBsAg+B7-H1组免疫后脾细胞产生的Th1类细胞因子(IFN-γ、IL-2)、HBsAg特异性分泌IFN-γ T细胞、T细胞增殖及血清HBsAb水平较对照组显著增加(P<0.05),但HBsAg组及HBsAg+B7-H1组之间无显著性差异.结论:HBsAg可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子,并诱导小鼠产生特异性的体液免疫,打破免疫耐受.B7-H1对HBsAg在HBV转基因小鼠中的免疫效果无影响.     

EB病毒潜伏膜蛋白2多表位的原核表达及其抗原特性分析

目的 对EB病毒(EBV)潜伏膜蛋白2(LMP2)中富含T、B细胞多表位肽段基因进行原核表达,并分析该多表位蛋白的抗原特性.方法 利用计算机在线软件预测EBV LMP2蛋白的CTL表位、Th表位.选取富含CTL表位和Th表位的肽段,兼顾其上下游已预测的B细胞表位,组成含多个T、B细胞表位的EBV LMP2多表位,该多表位基因序列经原核密码子优化后全序列合成,并克隆入原核表达载体pET32a(+)得到pET32a(+)/EBV-LMP2多表位重组质粒,经IPTG诱导在E.coli BL21(DE3)表达并纯化,经SDS-PAGE和Western blot分析鉴定;采用EBV膜蛋白家兔免疫血清和鼻咽癌患者血清进行Western blot分析其抗原特性;并利用EBV-LMP2多表位蛋白免疫BALB/c小鼠,分别采用LDH和ELISA方法检测小鼠脾细胞特异性CTL杀伤效应及血清特异性抗体IgG水平,以分析该表位蛋白的免疫原性.结果 LMP2(aa195-232)和LMP2(aa419-436)肽段富含CTL、Th和B细胞表位,将其串联后作为EBV LMP2多表位,该多表位基因在大肠杆菌中获得了表达.表达产物的相对分子质量(Mr)约27×103,与预期Mr相符;经Western blot证实EBV-LMP2多表位具有抗原特异性,可被EBV膜蛋白家兔免疫血清和鼻咽癌患者血清特异性抗体识别;小鼠免疫结果显示EBVLMP2多表位可诱导机体产生特异性的CTL杀伤效应,随着效靶比(1:5,1:10,1:25)增加,CTL杀伤活性逐渐增强(12.52%±2.59%,21.80%±1.08%,23.68%±3.74%),同时产生了特异性血清IgG抗体反应(A490=0.258±0.040),与对照组比较差异均具有统计学意义(P＜0.05).结论 本研究没计的EBV-LMP2多表位具有良好的抗原性和一定的免疫原性.     

重组减毒大肠杆菌对真核表达的CD8+ T细胞表位的运送研究

目的：分析体外、体内重组减毒大肠杆菌运送真核表达的CD8^＋T细胞表位的效应。方法：将携带融合表达绿色荧光蛋白（GFP）与OVACD8^＋T细胞表位基因真核表达质粒的重组大肠杆菌13A（pG2F）感染抗原提呈细胞（APC）IX^b和骨髓源树突状细胞（BMDC），应用体外抗原提呈试验检测APE对重组菌运送的CD8^＋T细胞表位的提呈效应。同时，重组菌13A（pG2F）以静脉注射方式免疫C57BL／6小鼠，应用ELISPOT法检测特异性IFN-γ分泌细胞。结果：感染试验表明，大肠杆菌13A能向APE中运送真核表达质粒，并且外源GFP基因获得了表达。体外抗原提呈试验结果显示，在感染的早期（2小时），LK^b和BMI）C均可提呈重组菌13A（pG2F）运送的T细胞表位；在感染的晚期（48小时），LK^b细胞对CD8^＋T细胞表位的提呈效应增强。在同样的作用条件下，BMI）C对减毒菌运送的抗原表位的提呈效应要强于LK^b细胞。体内结果显示，大肠杆菌可以有效运送真核表达的CD8^＋T细胞表位并诱导小鼠产生特异性细胞免疫应答。结论：重组减毒大肠杆菌在体外和体内均能有效运送真核表达的CD8^＋T细胞表位，为基于减毒细菌的新型基因工程疫苗的分子设计提供了有益借鉴。     

丙型肝炎病毒多表位抗原的原核表达及免疫原性分析

目的:研究丙型肝炎病毒(HCV)十表位抗原HCVe在大肠杆菌中的非融合表达及其免疫原性分析.方法:在大肠杆菌DH5α中表达非融合蛋白HCVe, 纯化该蛋白后利用Westerb blot分析其抗原反应性.免疫小鼠, 检测其免疫特异性.结果:HCVe十表位抗原能在原核表达系统中高效表达.Western blot和ELISA分析表明HCVe十表位抗原能与HCV病人阳性血清特异性结合, 并可诱发小鼠产生特异性HCV抗体.结论:表达的HCVe十表位抗原具有特异的抗原反应性及免疫原性.     

乙型肝炎病毒重组多表位抗原诱发的小鼠免疫应答

目的:对乙型肝炎病毒重组多表位抗原基因在大肠杆菌中表达的融合蛋白P/BPT进行免疫原性分析,以期为HBV预防/治疗性疫苗的研制奠定实验基础。方法:构建重组质粒pWR450-1/BPT,在大肠杆菌中诱导表达并纯化蛋白P/BPT,免疫BALB/c小鼠,乳酸脱氢酶释放法检测该融合蛋白诱导小鼠的细胞毒T淋巴细胞(CTL)应答,ELISA法检测小鼠血清抗-P/BPT抗体水平,流式细胞仪检测小鼠CD4+、CD8+T淋巴细胞亚群比例,用RT-PCR方法半定量检测细胞因子特异性mRNA,MTT法测淋巴细胞增殖效应。结果:P/BPT蛋白免疫后,效靶比为100:1时,可有效诱发特异性CTL应答(P<0.05);ELISA方法测出小鼠血清抗-P/BPTIgG明显升高(P<0.05);P/BPT蛋白免疫后,可显著刺激小鼠脾淋巴细胞增值(P<0.05);CD4+T淋巴细胞和细胞因子IFN-γ的增殖和分泌水平亦有明显升高。结论:该融合蛋白可有效诱导小鼠体液免疫和细胞免疫应答,为进一步研制乙肝预防治疗性疫苗奠定了初步基础。     

Rates of oxygen consumption and of anaerobic glycolysis in renal cortex and medulla of adult and new-born rats and guinea-pigs

1. Rates of oxygen uptake and of anaerobic glycolysis were estimated in slices from the renal cortex and medulla (a) of adult rats and guinea-pigs, (b) of new-born (1-, 5- and 21-day-old) rats and of guinea-pigs of 1, 12, 21, 24 and 120 hr age.2. In the adult rat, Q(O2) values for the cortex were 12.55 +/- 0.20 (22) and for the medulla: 8.56 +/- 0.17 (22) mul./hr.mg dry weight, while in the new-born rat (24 hr old) they were 10.99 +/- 0.46 (12) and 9.33 +/- 0.18 (9) mul./hr.mg dry weight respectively.3. Values for Q(CO2) (N2) (anaerobic glycolysis) in the 14 hr old new-born rat were in the renal cortex 9.65 +/- 0.35 (5) and in the medulla 7.39 +/- 0.43 (5) mul./hr.mg dry weight; while in the adult they were 2.25 +/- 0.08 (16) and 5.76 +/- 0.14 (16) mul./hr.mg dry weight, respectively.4. In the adult guinea-pig values for Q(CO2) (N2) were of the same order as in the adult rat, though the rate of O(2) uptake was for the cortex 8.12 +/- 0.22 (12) and for the medulla 5.02 +/- 0.23 (11) mul./hr.mg dry weight.5. Though the Q(O2) values in the renal cortex and medulla were smaller in the 1 hr old new-born guinea-pig, they were already increasing in the 12 hr old neonate.6. The results are discussed in the light of enzyme changes occurring during the process of maturation of the nephron as indicated by histochemical observations.     

Modes of reinforcement and gender and culture of experimenter and subject: effects of "alien" and external reinforcement for Japanese and American men and women

In this study, we examined external and "alien" reinforcement (ER and AR. respectively) as a factor in social learning, and studied the combined effects of culture and reinforcement mode. A female (Experiment 1) and a male (Experiment 2) experimenters conducted experimental sessions. Both men and women, who grew up in the same culture as the experimenter, participated and performed the experimental task. A three-way interaction effect of experimenter gender, culture, and reinforcement mode was found on task performance. And the effect was more pronounced for a Japanese experimenter. A female and a male experimenters conducted Experiments 3 and 4, respectively; however participants this time were men and women who grew up in different cultures than the experimenter. Results indicated that the pattern of the subject gender and reinforcement mode interaction effect, when the experimenter was Japanese with American subjects, was exactly opposite to that when the experimenter was American. These experiments showed that AR was as effective for social learning as ER, and that the cultural backgrounds of experimenter and subject influenced AR and ER effectiveness.     

Timing and intensity of early fevers and the development of allergies and asthma

BACKGROUND: Early childhood fevers appear to protect against later allergies and asthma. What is not known is the time in which fevers exert this effect and whether the degree of temperature increase is important. OBJECTIVE: We sought to examine the relationship between the time and degree of early fevers and later allergies and asthma. METHODS: Eight hundred thirty-five children from southeast Michigan were enrolled at birth. Clinic records from their first 2 years were abstracted for episodes of fever. At age 6 to 7 years, children underwent allergy testing. We examined fevers occurring within 6-month intervals in the first 2 years of life and outcomes at age 6 to 7 years. The primary outcome measures were allergic sensitization, asthma, asthma with allergic sensitization, and asthma without allergic sensitization. RESULTS: In the unadjusted analysis each episode of fever between 7 and 12 months of age was associated with a lower odds of allergic sensitization (odds ratio [OR], 0.71; 95% CI, 0.54-0.93) and asthma with allergic sensitization (OR, 0.43; 95% CI, 0.21-0.90) at age 6 to 7 years. Likewise, every 1 degrees C increase in the maximum temperature between 7 and 12 months was associated with a lower odds of allergic sensitization (OR, 0.77; 95% CI, 0.61-0.96) and asthma with allergic sensitization (OR, 0.62; 95% CI, 0.40-0.94). After adjusting for potential confounders, each episode of fever between 7 and 12 months was associated with a lower likelihood of asthma with allergic sensitization (adjusted OR, 0.33; 95% CI, 0.11-0.94) at age 6 to 7 years. CONCLUSIONS: Both the timing and intensity of childhood fevers appear to be important factors in the development of allergies and asthma.     

纳米水平分子成像与诊疗一体化研究进展与挑战

分子成像能以非侵入性的方式重现活体细胞的生理功能和生物学过程,提高疾病的早期和特异性诊断水平。纳米颗粒/材料具有物理性质可控性高、易于表面修饰、血液循环时间长和可功能化等优点,在疾病诊断与治疗中显示出巨大潜力。但如何阐明纳米材料多功能间的内在联系、解决其代谢及安全性等关键机制难题、实现纳米颗粒/材料多功能性到临床多功能...