鼠疫菌质粒上标识基因的筛选

目的 筛选鼠疫菌质粒上保守、稳定、特异的DNA标识序列.方法 选择32条源于鼠疫菌质粒上DNA序列,采用常规PCR技术,对云南家、野鼠两型共40株鼠疫菌、47株非鼠疫菌、鼠疫菌疫苗株EV76(阳性对照),进行了特异性验证试验和稳定性鉴定试验.结果 筛选出4对相对保守、稳定、特异的DNA标识序列:YPMT1.05c,YPMT1.03c,YPMT1.42及YPMT1.04c.结论 所筛选的4对鼠疫菌DNA标识序列,可用于鼠疫快速诊断.     

鼠疫菌感染兔血清抗体谱分析

目的分析鼠疫耶尔森菌感染的兔血清(鼠疫菌感染兔血清)中抗体产生的种类与规律,为了解鼠疫菌的致病机制及筛选新的疫苗亚单位提供实验依据。方法利用含有145个鼠疫菌毒力相关蛋白的蛋白质芯片检测鼠疫菌感染兔血清抗体谱,并与鼠疫菌活疫苗EV76株免疫兔血清(免疫兔血清)抗体谱比较。结果在鼠疫菌感染兔血清中共检测到41个蛋白相应的抗体,其中28个抗体在免疫兔血清中也检测到,有6个蛋白抗体只在鼠疫菌感染兔血清中检测到。结论鼠疫菌感染兔血清的抗体谱与免疫兔血清的抗体谱不完全一致,通过对鼠疫菌感染兔血清抗体谱的研究,可为深入了解细菌的致病机制及筛选新的疫苗亚单位奠定基础。     

HBV和HCV核酸疫苗的临床应用前景

核酸疫苗是指将含有编码抗原蛋白的DNA序列及必要的表达调控元件的重组质粒DNA直接导入机体组织，通过宿主细胞的转译系统表达目的抗原，诱导机体发生免疫应答的一种新型疫苗。近年来核酸疫苗的研究发展迅速，在乙型肝炎和丙型肝炎防治方面也取得了令人瞩目的成绩。1. HBV、HCV核酸疫苗的目的基因和载体：HBV有多种抗原蛋白编码基因。包括编码包膜蛋白的S基因、preS1基因、preS2基因，编码HBcAg和HBeAg的Ｃ基因等。包膜蛋白能诱导保护性抗体；HBcAg是CTL的靶抗原，其诱发的CTL应答有利于清除HBV，而HBeAg诱导抗HBe产生并减…     

青海省鼠疫患者血清抗体谱研究

目的研究在鼠疫感染过程中,患者对鼠疫耶尔森菌产生体液免疫反应的规律.寻找鼠疫菌新的具有免疫原性的蛋白。方法利用鼠疫耶尔森菌重要毒力相关蛋白芯片对鼠疫患者的血清抗体谱进行检测。结果利用含145个鼠疫耶尔森菌蛋白质的蛋白芯片。共检测到鼠疫患者血清中29个蛋白的抗体。其中13种蛋白的抗体在患者体内明显上升,除已知的8个具有免疫原性的蛋白外,发现了鼠疫菌5个新的具有免疫原性的蛋白(SycE、SycH、OmpA、pH6与YPO2098)。结论这5个新的具有免疫原性的蛋白将是下一步鼠疫疫苗研究的靶点。     

蛋白质芯片在鼠疫患者血清抗体谱检测中的应用

目的检测鼠疫患者血清抗体谱,为鼠疫的疫苗研究奠定基础。方法利用鼠疫耶尔森氏菌毒流力相关蛋白的蛋白质芯片检测鼠疫患者血清抗体谱。结果利用一个包含145个鼠疫菌毒力相关蛋白的蛋白质芯片,在鼠疫患者血清中检测到37种鼠疫菌蛋白相应的抗体。结论通过对鼠疫患者血清抗体谱的分析,寻找到新的能在人体内产生体液免疫的蛋白。     

刚地弓形虫ROP2-P30复合重组蛋白疫苗对BALB／c小鼠免疫保护性的研究

目的用刚地弓形虫ROP2-P30复合重组蛋白疫苗免疫BALB/c小鼠观察对机体的影响,为研制安全有效的弓形虫疫苗奠定基础。方法PCR方法从刚地弓形虫基因组中扩增出ROP2和P30片段,将这两个片段分别亚克隆至pET-30a原核表达载体中,表达出ROP2-P30复合重组蛋白。用蛋白疫苗免疫BALB/c小鼠,ELISA检测免疫小鼠后体液中抗体和细胞因子的变化,观察攻击试验小鼠的的死亡率。结果ROP2-P30复合重组蛋白疫苗免疫BALB/c小鼠诱导机体产生大量的IgM、IgG抗体和IFN-γ、IL-2及IL-4细胞因子。攻击试验表明,复合重组蛋白免疫组和对照组相比,小鼠的存活时间明显延长。结论ROP2-P30复合重组蛋白免疫BALB/c小鼠诱导其产生高水平的体液和细胞免疫应答,该复合重组蛋白是抗刚地弓形虫感染的候选疫苗之一。     

日本血吸虫Rho GTPase DNA疫苗及重组蛋白疫苗免疫保护机制初探

目的　初步探讨日本血吸虫中国大陆株pcDNA3 .1 SjRhoGTPaseDNA疫苗、重组蛋白疫苗以及联合疫苗免疫的保护机制。　方法　将 pcDNA3 .1 SjRhoGTPaseDNA疫苗、重组蛋白疫苗分别及联合疫苗免疫昆明鼠后 ,分不同时间采集血清 ,以ELISA法测定总抗体及IgG亚类。　 结果　重组蛋白疫苗及联合疫苗诱导产生的抗体效价较高 ,DNA疫苗及联合疫苗诱导产生的抗体以IgG2a为主 ,重组蛋白疫苗诱导产生的抗体以IgG1为主。　 结论　Sj RhoGTPase基因DNA疫苗及联合疫苗主要诱生Th1型细胞免疫应答 ,重组蛋白疫苗主要诱生Th2型体液免疫应答     

日本血吸虫Rho GTPase DNA疫苗及重组蛋白疫苗免疫保护机制初探

目的 初步探讨日本血吸虫中国大陆株pcDNA3．1-SjRho GTPase DNA疫苗、重组蛋白疫苗以及联合疫苗免疫的保护机制。方法 将pcDNA3．1-SjRho GTPase DNA疫苗、重组蛋白疫苗分别及联合疫苗免疫昆明鼠后，分不同时间采集血清，以ELISA法测定总抗体及IgG亚类。结果 重组蛋白疫苗及联合疫苗诱导产生的抗体效价较高，DNA疫苗及联合疫苗诱导产生的抗体以IgG2a为主，重组蛋白疫苗诱导产生的抗体以IgG1为主。结论 Sj-Rho GTPase基因DNA疫苗及联合疫苗主要诱生Th1型细胞免疫应答，重组蛋白疫苗主要诱生Th2型体液免疫应答。     

幽门螺杆菌ureI核酸疫苗免疫活性的初步研究

目的 观察幽门螺杆菌ureI核酸疫苗诱导C57BL/6小鼠产生体液免疫应答及细胞免疫应答水平.方法 重组质粒pcDNA3.1(+)/ureI、空质粒pcDNA3.1(+)及PBS分别肌注免疫C57BL/6小鼠.ELISA法检测小鼠血清中特异性IgG抗体水平以及脾淋巴细胞培养上清中IL-4和IFN-γ含量,MTT比色法检测脾淋巴细胞增殖反应,PCR和免疫荧光组化法检测ureI基因和蛋白表达.结果 pcDNA3.1(+)/ureI能够诱导小鼠产生特异性IgG抗体,该组小鼠脾淋巴细胞经UreI重组蛋白刺激后,其刺激指数和培养上清中IL-4、IFN-γ含量明显升高; ureI基因可在小鼠肌细胞中有效表达.结论 pcDNA3.1(+)/ureI核酸疫苗能刺激C57BL/6小鼠产生较强的体液免疫应答和细胞免疫应答,为进一步研究该疫苗的免疫保护作用奠定了基础.     

乙型肝炎病毒核心抗原与表面抗原"a"决定簇融合疫苗的免疫效果

目的 评价乙型肝炎病毒核心蛋白作为类病毒颗粒载体融合表面抗原"a"决定簇的基因疫苗和蛋白疫苗的免疫效果,为寻求有效的慢性乙型肝炎治疗性疫苗奠定基础.方法 体外分别扩增编码乙型肝炎病毒HBsAg 100～162 aa部分、HBcAg 1～78 aa部分和HBcAg 83～144aa的基因序列,目的 基因连接后定向克隆到真核表达载体pcDNA3的多克隆位点内.将嵌合基因亚克隆到原核表达载体,诱导表达并纯化获得融合蛋白.分别用真核表达质粒和纯化融合蛋白免疫BALB/c小鼠,加强免疫并定期采血,检测血清中特异性抗体并进行淋巴细胞增殖实验以评价体液和细胞免疫应答效果.实验结果进行重复测量资料的方差分析和单向方差分析. 结果嵌合基因免疫后可以产生有效的抗-HBs"体液免疫"应答,免疫0、2、4、6、8、10、12,14周时的S/N值分别为1.05±0.20、2.69±0.38、4.48±0.50、6.61±0.49、6.95±0.19、7.43±0.31、8.11±0.14、7.43±0.31,F值分别为1202.021和200.059,P<0.01.未引起明显的抗-HBc"体液免疫"应答却获得了明显的HBcAg特异性细胞免疫应答.融合蛋白免疫后也能获得有效的抗-HBs"体液免疫"应答,但针对HacAg的体液应答却较强;可以产生较强的HBcAg特异性增殖反应,而没有明显的HBsAg特异性增殖反应.嵌合基因免疫组较融合蛋白组的细胞免疫应答明显增强,体液免疫应答相对较弱. 结论 以该乙型肝炎病毒核心蛋白为载体的包含HBsAg"a"决定簇的融合基因疫苗相对于其融合蛋白具有良好的细胞和体液免疫效果.     

Serologic survey of the sentinel animals for plague surveillance and screening for complementary diagnostic markers to F1 antigen by protein microarray

Plague is a deadly infectious disease caused by the gram-negative bacterium, Yersinia pestis. In 2005, five plague patients were confirmed in the Yulong County of the Yunnan Province, China. In this study, the serologic survey of > 2,900 serum samples from domestic dogs and cats in and around the county, where human plague occurred, confirmed that domestic dogs and cats could serve as sentinel animals for plague surveillance. Meanwhile, the antibody responses in the infected dogs and cats were profiled by microarray containing 218 proteins of Y. pestis. In addition to F1, LcrV, YPCD1.28c, and YPO2118 induced humoral responses in all or most of the individuals, providing complementary candidates to F1 antigen for diagnostic markers of plague.     

腺鼠疫患者血清抗体谱的研究

目的检测腺鼠疫患者血清抗体谱及抗体随时间变化趋势。方法利用一包含145个鼠疫耶尔森氏菌毒力相关蛋白质的蛋白芯片检测云南腺鼠疫患者血清抗体谱及抗体随时间变化趋势。结果在腺鼠疫患者体内检测到32种蛋白相应的抗体。结论FI抗体产生的速度最快、幅度最高、持续的时间最长;YopM、YopH、YopE抗体升高不明显;V抗体在患者体内未检测到;在发病后14 d才检测到pH6抗原的抗体,3个月时抗体荧光值没有下降,可持续半年。YopD的抗体在部分患者体内明显升高,但在半年后下降到正常水平。     

Recombinant capsular antigen (fraction 1) from Yersinia pestis induces a protective antibody response in BALB/c mice

Yersinia pestis produces a glycoprotein capsule, the biosynthesis of which appears to be temperature dependent. The fraction I (F1) component of this capsule is specific to Y. pestis and the detection of F1 antibodies is the basis for several serological tests. We report the cloning of the F1 gene and its expression in Escherichia coli using the phagemid vector lambda ZAPII and a F1-specific monoclonal antibody. The recombinant F1 antigen had a molecular weight of 17 kDa, which proved to be identical to that of the F1 antigen produced by Y. pestis. The recombinant cells produced F1 antigen at 37 degrees C but only minimal amounts at 27 degrees C, suggesting that the genetic features affected by temperature in Y. pestis may be operating in the E. coli clone. It is not known if their similar molecular weights reflect the glycosylated nature of both proteins. F1 antigen purified from the E. coli recombinant induced a protective immune response in BALB/c mice challenged with up to 10(5) virulent Y. pestis. The resistance of immunized mice to plague infection correlated with high titers of F1 antibody. The cloned gene expresses an immunogenically competent F1 antigen suitable for use in plague serodiagnostics and vaccine development.     

Immunity in plague: protection of the vervet (Cercopithecus aethips) against pneumonic plague by the oral administration of live attenuated Yersinia pestis.

Protection against pneumonic plague by the oral administration of a live, attenuated Yersinia pestis vaccine, EV76 (Paris) F, was evaluated in the vervet (Cercopithecus aethips). Six animals were vaccinated with a dose of 1.175 X 10(9) colony-forming units (cfu); all tolerated the dose and developed antibodies to Fraction I of Y. pestis. Three immunized animals were challenged with an inhaled dose of 3.2 X 10(6) cfu (160 LD50 [50% lethal dose]) of virulent Y. pestis strain 195/P and two survived; the other three received a challenge dose of 4.9 X 10(6) cfu (245 LD50) and one survived. The three surviving monkeys had high titers of antibody to Fraction I. At necropsy of the animals that succumbed to the challenge infection, almost complete consolidation of the lungs was apparent, and sizable effusions were present in the thoracic cavities. Indeed, the striking pathology indicated the severest form of pneumonic plague. Since it can be assumed that the animal surviving challenge with the larger inoculum would have survived the smaller dose, we conclude that the immunization procedure protected half of the vervets that were exposed to challenge.     

对两例隐性感染鼠疫的分析报告

本文分析了在人类鼠疫流行时发生的隐性感染鼠疫两例。一例系接触病獭,一例与肺鼠疫患者直接接触所致。虽未分离到鼠疫菌,但通过间接血凝证实确系鼠疫菌感染。这可能与侵入机体的菌量、鼠疫菌毒力、机体反应性以及接种鼠疫活菌苗有关。对隐性感染鼠疫在人类鼠疫流行时应予以注意。     

从接触者抗体水平看鼠疫的传染性

目的检测接触者抗体水平看鼠疫的传染性。方法用IHA检测1例败血型鼠疫患者和1例腺鼠疫的密切接触者血清抗体。结果共检出3份阳性血清。结论2例鼠疫患者均有早期肺部感染的症状,推测密切接触者血清抗体的出现系由鼠疫感染所致,提示腺鼠疫和败血型鼠疫具有很高的继发肺鼠疫的倾向,在疫情处理过程中应引起足够重视。     

Protective immunity against respiratory tract challenge with Yersinia pestis in mice immunized with an adenovirus-based vaccine vector expressing V antigen

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine.