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1.
本文采用超声破碎TritonX—100和超速离心技术提取了鼠伤寒杆菌(Salmonellatyphimurium,STM)的外膜蛋白(Outermembraneproteins,OMPs),并发现OMPs中脂多糖LPS的含量约为5%,OMPs经SDS—PAGE显示10余条蛋白带。对OMPs诱发BALB/C小鼠产生典型迟发型变态反应DTH和IL—2的水平进行了检测。经腹腔免疫的小鼠用500LD50鼠伤寒杆菌(50115)攻击,100%可得到保护;用500LD50伤寒杆菌(E686)攻击,33.3%可得到交叉保护。免疫BALB/C小鼠的T淋巴细胞,经尾静脉注射给非免疫小鼠,可使后者得到85.7%的被动免疫保护,上述结果说明OMPs能诱发BALB/C小鼠细胞免疫和保护性免疫,并提示成为分子疫苗的可能性。  相似文献   

2.
本实验采用超声破碎、TritonX-100处理和超速离心技术提取了鼠伤寒沙门氏菌(Salmonellatyhimurium,STM)的外膜蛋白(Outermembraneproteins,OMPs),其中脂多糖(LPS)的含量约为5%。OMPs经SDS—PAGE显示10余条蛋白带。对OMPs诱发BALB/C小鼠产生典型的迟发型变态反应(DTH)进行了检测。经腹腔免疫的BALB/C小鼠用500LD50鼠伤寒沙门氏菌(50115)攻击,100%可得到保护;用500LD50伤寒杆菌(E686)攻击  相似文献   

3.
为了阐明肾综合征出血热纯化疫苗(I型)中糖蛋白(GP)、核蛋白(NP)在免疫保护中的作用,采用脾细胞转输法,分别将GP、NP、GP+ NP免疫小鼠后的脾细胞与未经免疫的小鼠正常脾细胞(3.5×105 个)经腹腔转输给乳鼠,2 h 后以100 LD50的汉滩(HTN)病毒(LR1 株)进行脑腔攻击。结果表明,其乳鼠的保护率分别达77.0% ,38.5% 和92.3% ,而未经免疫的小鼠正常脾细胞则不能提供保护(0% ),揭示了GP+ NP在抗HTN 病毒感染中具有免疫协同作用。  相似文献   

4.
Metylomonassp.GYJ3菌的甲烷单加氧酶(MMO)粗酶提取液经DEAE-SepharoseCL-6B阴离子交换层析、SephadexG-100凝胶过滤层析和DEAE-TSKgelHPLC分离纯化出MMO还原酶组分.经HPLC分析,纯度大于95%,纯化倍数为4.4,加入至MMO羟基化酶和调节蛋白B的体系中表现比活为228nmol环氧丙烷每分钟毫克蛋白.SDS-PAGE电泳表明还原酶由一种亚基组成,分子量42kD.ICP-AES测定还原酶的Fe含量为1.83molFe每mol蛋白.UV-Vis光谱表明还原酶除280nm蛋白质特征峰外在460nm有最大吸收峰,且A280nm/A460nm为2.50,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个FAD辅基和Fe2S2中心.在厌氧条件下,还原酶能够和NADH作用,UV-Vis光谱分析表明还原酶460nm处特征吸收峰消失,说明在MMO催化过程中还原酶接受NADH的电子.DEAE-SepharoseCL-6B阴离子交换层析分离出调节蛋白B,部分纯化的调节蛋白B的分子量大约在20kD,它能够提高MMO比活性40倍,MMO还原酶和调节蛋白B单独存在时不具有MMO  相似文献   

5.
长白山白眉蝮蛇蛇毒磷脂酶A2的分离和初步表征   总被引:10,自引:0,他引:10  
东北长白山白眉蝮蛇(AgkistrodonblomhoffiUsurensis)蛇毒经DEAESephadexA50离子交换层析柱,连续3步SephadexG75凝胶过滤柱得到了磷脂酶A2(PLA2)的纯品。SDS聚丙烯酰胺凝胶电泳(SDSPAGE)以及基质辅助激光解析电离飞行时质谱(MALDI/TOF/MS)表征为单一蛋白,其准确分子量为(14.008±0.007)kD。最适pH范围8.0~9.0,最适的反应温度为45℃。在溶液中有多聚体的存在。  相似文献   

6.
用正丁醇抽提,硫酸铵分级沉淀,DEAE-纤维素和SephacrylS-200柱层析,从南方鲇(Silurus meridionalis Chen)肠粘膜中提取出碱性磷酸酶(AKP)。提纯倍数为39.50倍,比活为68.35μ/mg蛋白,提取酶液经PAGE和SDS-PAGE只呈现一条区带。该酶的分子量为132140,N末端氨基酸为门冬氨酸,最适pH为10.10,7.5>pH>11.5时不稳定,最适温度为40℃左右,对热不很稳定,以磷酸苯二钠为底物其K_m值为1.72×10~(-3)mol/L。Mg~(2+)、Mn~(2+)为该酶的激活剂,KH_2PO_4、L-CyS、ME、DFP、EDTA-Na_2为抑制剂。选用KH_2PO_4和DFP作抑制类型的判断,结果表明,KH_2PO_4属竞争性掏剂,其抑制常数为2.3mmol/L;DFP为非竞争性抑制剂,抑制常数为1.05mmol/L。  相似文献   

7.
动脉平滑肌细胞(sm ooth m uscle cell,SMC)是动脉粥样硬化(atherosclerosis,AS)斑块中的主要细胞,它的增殖在AS形成过程中极其重要.利用体外培养的人主动脉SMC,观察了天然高密度脂蛋白(native high density lipoprotein,N-HDL)及氧化修饰HDL(oxidized HDL,OX-HDL)对培养人主动脉SMC cyclin D1(细胞周期蛋白D1)基因转录表达的影响.结果表明:(1)N-HDL对SMCcyclin D1基因表达无影响(P> 0.05);(2)OX-HDL使SMCcyclin D1基因表达显著增强(P<0.01),其表达量随时间(2、12、24 h)延长而增加.上述结果表明,OX-HDL的致AS作用可能与其刺激SMCcyclin D1基因表达增加有关.  相似文献   

8.
低温胁迫下红松幼苗活性氧的产生及保护酶的变化   总被引:80,自引:0,他引:80  
在不同低温胁迫时间下,对红松(Pinus koraiensis Sieb.et.Zucc)幼苗针叶中H2O2、O^-.2、膜脂过氧化产物丙二醛(MDA)、组织自动化氧化速率及保护酶超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(ASP)的动态变化过程进行了测定。结果表明,随着低温胁迫时间的延长,O^-.2产生速率和H2O2含量先上升后下降;MDA的含量呈波  相似文献   

9.
MPEG-SOD对急性低氧下鼠左心功能的保护作用   总被引:1,自引:0,他引:1  
单甲氧基聚乙二醇(MPEG)活化后与超氧化物歧化酶(SOD)偶联,经纯化后得到MPEG-SOD,鉴定其分子量约为7.0×105Dation。其在血液循环中酶活性半衰期超过30h,远大于天然SOD(6-10min)。SD雄性大鼠分三组.对照组(n=8.由股静脉注入0.2ml生理盐水)、NativeSOD组(n=8,注以0.2ml生理盐水配的800USOD)和MPEG-SOD组(n=9,注以800UMPEG-SOD)。低氧前三组鼠的心率(HR)、左室内峰压(LVP)、左室压微分(LV±dp/dtmax)和股动脉压(AP)均无统计学差别。在模拟6000—6500m高度急性低氧1.8.14min记录上述各项指标,结果如下:除HR外,NativeSOD组与对照组各指标均无统计学差别,MPEG-SOD组的各项指标均明显高于对照组。表明MPEG-SOD对急性低氧导致的左心室力学指标的减弱有明显保护作用,提示超氧自由基(O2-)在急性低氧导致左心室功能损伤中起重要作用,即可能是由O2-及其衍生的其它自由基损害心肌细胞生物膜系统所致。  相似文献   

10.
T—2毒素对心肌细胞电生理特性的影响及硒的保护作用   总被引:3,自引:0,他引:3  
本实验在培养的Wistar大鼠乳鼠心肌细胞上观察了T-2毒素对膜电位活动的影响及硒的保护作用。结果表明,T-2毒素(0.1,0.5,5.0mg/L)使心肌细胞动作电位幅值(APA)、超射(OS)、阈电位(TP)、最大舒张电位(MDP)及最大除极速率(Vmax)显著降低。使复极化时程(APD10、APD50、APK90)延长;动作电位发放频率(APF)受到明显抑制。提示T-2毒素能抑制Ca^2+、K  相似文献   

11.
Protective effect of immunization with heat shock protein (HSP) against bacterial and viral infections in mice was studied. Recombinant HSP 70 kDa of Mycobacterium tuberculosis contaminated with lypopolysaccharide (0.185 mcg/ml) was used for experiments. One intraperitoneal injection of 100 or 400 mcg of HSP induced rapid protection against intraperitoneal challen e with 125 LD50 of Salmonella typhimurium (on 3rd-6th day) and against intranasal challenge with 10 LD50 of avirulent for humans avian influenza virus H5N2 (A/ mallard/Pennsylvania/10218/84) (on 5th-8th day). Three daily injections with 10 mcg of HSP induced rapid, significant and long-term protection against S. typhimurium. Immunization with HSP protected 100% of mice during 3 days after the challenge, 50% of immunized animals survived during 21 days (duration of the study). All nonimmunized mice died on 6th day.  相似文献   

12.
The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.  相似文献   

13.
The 43-kDa non-O antigenic component isolated from the crude ribosomal fraction of Salmonella typhimurium [9] was further purified by affinity chromatography (43-kDa protein: 43-kDp). Immunization with 43-kDp did not induce complete mouse protection in CF1 mice to 500 LD50 of S. typhimurium, although it elicited a substantial IgG antibody response. The 43-kDp exhibited the mitogenicity to splenocytes (CF1 and C3H/HeJ) and B cell-rich populations (CF1). Complexing 43-kDp with the compact ribosomes of Streptococcus pyogenes by formaldehyde (complex vaccine: CV) elicited both IgM and IgG antibodies to 43-kDp. CV induced a boosting effect to enhance IgG antibody response. Moreover, CV generated delayed-type hypersensitivity to salmonella antigens and also conferred complete protection against 500 LD50 challenge of S. typhimurium to CF1 mice. These abilities of CV were reduced or impaired by RNase digestion. CV was able to induce partial or complete protection in inbred mouse strains (C3H/HeN, C3H/HeJ, DBA/2 and A/J). These data, in addition to other reports, suggest that conformational stability between ribosomes and contaminating substances such as 43-kDp or O-antigens might be required for the overall effects of the ribosomal vaccine.  相似文献   

14.
Protective immunity induced by porin in experimental mouse salmonellosis   总被引:7,自引:0,他引:7  
The induction of protective immunity to mouse salmonellosis by porin from Salmonella typhimurium LT2 was studied. The immunization with porin induced a high level of protective immunity to salmonellosis in BALB/c mice. Mice immunized with porin exhibited significant levels of delayed-type hypersensitivity response and interleukin-2 production, indicating that porin was capable of inducing cell-mediated immunity (CMI). Furthermore, we found that both T cells and sera taken from the porin-immunized mice could transfer the protection against salmonellosis into nonimmunized mice. These observations suggested that a high level of the protection to salmonellosis obtained by the porin immunization resulted from the induction of CMI in addition to humoral immunity.  相似文献   

15.
Zhao Z  Wakita T  Yasui K 《Journal of virology》2003,77(7):4248-4260
We established a simple and effective method for DNA immunization against Japanese encephalitis virus (JEV) infection with plasmids encoding the viral PrM and E proteins and colloidal gold. Inoculation of plasmids mixed with colloidal gold induced the production of specific anti-JEV antibodies and a protective response against JEV challenge in BALB/c mice. When we compared the efficacy of different inoculation routes, the intravenous and intradermal inoculation routes were found to elicit stronger and more sustained neutralizing immune responses than intramuscular or intraperitoneal injection. After being inoculated twice, mice were found to resist challenge with 100,000 times the 50% lethal dose (LD(50)) of JEV (Beijing-1 strain) even when immunized with a relatively small dose of 0.5 micro g of plasmid DNA. Protective passive immunity was also observed in SCID mice following transfer of splenocytes or serum from plasmid DNA- and colloidal gold-immunized BALB/c mice. The SCID mice resisted challenge with 100 times the LD(50) of JEV. Analysis of histological sections detected expression of proteins encoded by plasmid DNA in the tissues of intravenously, intradermally, and intramuscularly inoculated mice 3 days after inoculation. DNA immunization with colloidal gold elicited encoded protein expression in splenocytes and might enhance immune responses in intravenously inoculated mice. This approach could be exploited to develop a novel DNA vaccine.  相似文献   

16.
The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.  相似文献   

17.
Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium. The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E. multilocularis eggs was evaluated. Oral or intraperitoneal immunisation with live attenuated S. typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E. multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8%. The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis. By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis antibodies were detectable in the sera, immunisation with E. coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections. These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.  相似文献   

18.
The ability of a newly identified probiotic lactic acid bacterial strain, Bifidobacterium lactis (HN019), to confer protection against Salmonella typhimurium was investigated in BALB/c mice. Feeding mice with B. lactis conferred a significant degree of protection against single or multiple oral challenge with virulent S. typhimurium, in comparison to control mice that did not receive B. lactis. Protection included a ten-fold increase in survival rate, significantly higher post-challenge food intake and weight gain, and reduced pathogen translocation to visceral tissues (spleen and liver). Furthermore, the degree of pathogen translocation showed a significant inverse correlation with splenic lymphocyte proliferative responses to mitogens, blood and peritoneal cell phagocytic activity and intestinal mucosal anti-S. typhimurium antibody titers in infected mice; all of these immune parameters were enhanced in mice fed B. lactis. Together, these results suggest that dietary B. lactis can provide a significant degree of protection against Salmonella infection by enhancing various parameters of immune function that are relevant to the immunological control of salmonellosis. Thus dietary supplementation with B. lactis provides a unique opportunity for developing immune-enhancing probiotic dairy food products with proven health benefits.  相似文献   

19.
[目的]通过建立的小鼠呼吸道感染模型评价重组百日咳杆菌黏附素蛋白(GST-PRN)对小鼠的免疫保护效力.[方法和结果]在主动免疫保护试验中,GST-PRN免疫组小鼠能产生较高的PRN抗体水平,在使用3xLD50的支气管败血波氏杆菌HH0809株进行呼吸道气雾攻毒后,其保护率为100%(20/20),但载体蛋白GST和PBS对照组小鼠的存活率仅为15%(3/20)和20%(4/20).在被动免疫保护试验中,腹腔免疫GST-PRN兔抗血清能100%(10/10)保护小鼠抵抗10×LD50的HH0809株的腹腔攻击,但GST兔抗血清和PBS免疫组小鼠的存活率均为0(0/10和0/9).[结论]研究结表明重组PRN蛋白具有良好的免疫学活性,可作为亚单位疫苗或疫苗添加成分.  相似文献   

20.
A vaccine prepared from the residue after extraction of whole cells of Salmonella typhimurium with 2% sodium deoxycholate proved to be nontoxic and highly immunogenic. The material was not lethal for mice at 6.0 mg and was essentially nontoxic in rabbit skin, whereas endotoxic activity was found in the dialyzed extract. A high dosage, above 2.0 mg, was less protective than lower doses, indicating a degree of "immunologic paralysis." Three inoculations of low doses, 0.25 mg each, induced protection against death and tissue infection in animals challenged with 2,000 ld(50) of virulent homologous S. typhimurium and against death, but not against tissue infection, after heterologous challenge with S. enteritidis. Residues of purified cell walls were as effective as residues of whole cells, indicating that the immunizing antigen(s) resided in the cell wall.  相似文献   

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