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1.
Purpose: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB) protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs) in Escherichia coli BL21 (DE3). Materials and Methods: The ctxB fragment was amplified from Vibrio cholerae O1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F’ and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4). The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3) cells to express CTB protein. Result: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. Conclusion: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.  相似文献   
2.
目的 构建表达幽门螺杆菌融合蛋白粘附素-细胞空泡毒素A-霍乱毒素B亚单位(HpaA-CtxB-VacA,HCTV)的原核表达载体。并诱导表达,为制备具有治疗与预防作用的多联疫苗奠定基础。方法用PCR技术从pQE-hctB扩增hpaA和ctxB目的基因片段。从pQE30-V质粒扩增出vacA基因,同时插入表达载体pQE-30,构建成pQE-hctv。再将poE-hctv转化大肠杆菌DH5α,经IPTG诱导表达。SDS-PAGE分析表达结果。Western blotting鉴定其抗原性。结果融合蛋白的相对分子量约为68000,可溶性表达占全菌的15%以上,经亲和层析后可获得纯度为90%以上的重组蛋白。Western blotting分析其能分别与HpaA抗血清、VacA抗血清和CT抗血清特异性反应。结论重组表达质粒pQE30-hctv表达成功,而且具有各自蛋白的抗原性,为进一步研究融和蛋白的功能和制备集预防和治疗为一体的Hp候选口服疫苗提供基础。  相似文献   
3.
Objectives: To examine eight strains of Vibrio cholerae O1 isolated in 2003 and 2005 from Puri, India, for antibiotic susceptibility, presence of virulence and regulatory genes, cholera toxin (CT) production, CTX arrangement and genomic profiles. Materials and Methods: Bacterial strains were tested for antibiotic susceptibility using disc diffusion assay. Polymerase chain reaction determined the presence of antibiotic resistance, virulence and regulatory genes. To determine the type of cholera toxin subunit B (ctxB), nucleotide sequencing was performed. Southern hybridisation determined the number and arrangement of CTXΦ. Ribotyping and pulsed-field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. Results: All the eight strains, except one strain, showed resistant to nalidixic acid, sulphamethoxazole, streptomycin and trimethoprim and possessed the sullI, strB, dfrA1 and intSXT genes. All the strains carried the toxin-co-regulated pilus pathogenicity island, the CTX genetic element, the repeat in toxin and produced CT. Restriction fragment length polymorphism (RFLP) analysis showed that V. cholerae O1 possess a single copy of the CTX element flanked by tandemly arranged RS element. Nucleotide sequencing of the ctxB gene showed the presence of classical ctxB. RFLP analysis of conserved rRNA gene showed two ribotype patterns. PFGE analysis also showed at least three PFGE patterns, irrespective of year of isolations, indicating the genomic relatedness among them. Conclusion: Overall, these data suggest that classical ctxB-positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is need to continuous monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.  相似文献   
4.
《Vaccine》2015,33(15):1880-1889
Live attenuated vaccines are cost effective approach for preventing a broad range of infectious diseases, and thus are of great interest. However, immune-defects can predispose the patient to infections by the vaccine candidate itself. So far, few live vaccine candidates have been designed specifically for immune compromised individuals. Recently, we reported a new Salmonella Typhimurium Z234-vaccine strain (Periaswamy et al., PLoS ONE 2012;7:e45433), which was specifically attenuated in the NADPH-oxidase deficient host. In the present study, the Z234-vaccine strain was further engineered to express heterologous antigen (Vibrio cholerae toxin antigen subunit-B, i.e. CtxB) with the intention of creating a vector for simultaneous protection against Cholera and Salmonellosis. The primary aim of this study was to ensure the expression of CtxB antigen by the recombinant vaccine strain Z234-pMS101. The antigen CtxB was expressed through Z234 as a fusion protein with N-terminal signal sequence of Salmonella outer protein (SopE), an effector protein from Salmonella under the control of SopE promoter. The CtxB-expressing plasmid construct pMS101 (pM968-pSopE-ctxB) was found to be stable both in vitro and in vivo. In an oral mouse infection model, the vaccine strain Z234-pMS101 efficiently colonized the host gut. The extent of protection was confirmed after challenging the immunized hosts with live V. cholerae. Vaccinated mice showed reduced gut colonization by V. cholerae. Further assessment of immunological parameters supported the possibility of conferring effective immune response by Z234-pMS101 vaccine strain. Overall, the Z234-pMS101 vaccine strain showed potential as a promising polyvalent vaccine candidate to protect against S. Typhimurium and V. cholerae infection simultaneously.  相似文献   
5.
目的 构建霍乱毒素B亚单位编码基因(ctxB)的真核表达质粒pVAX1-ctxB,并观察其在真核细胞中的蛋白表达情况.方法 利用基因重组技术构建真核表达质粒pVAX1 -ctxB,通过脂质将其转染至CHO细胞中诱导蛋白表达,GM1 -ELISA法检测其蛋白表达产物.结果 重组质粒pVAX1-ctxB经酶切可得到与ctxB大小相同的片段,GM1 -ELISA法证实经转染的CHO细胞可分泌霍乱毒素B亚单位(ctxB)蛋白.结论 成功构建重组表达质粒pVAX1- ctxB,其蛋白表达产物具有ctxB蛋白活性,为下一步加强基因防龋疫苗效价奠定了基础.  相似文献   
6.
中国O1群El Tor霍乱弧菌产毒株表型多态性研究   总被引:1,自引:0,他引:1       下载免费PDF全文
目的 了解近50年中国不同地区分离的O1群El Tor型霍乱弧菌产毒株的生物表型特征变化。方法 采用多粘菌素B敏感试验、第Ⅳ组霍乱噬菌体裂解试验、VP试验和溶血实验进行表型特征分析。结果 生物型表型特征分析表明133株菌具有典型的El Tor生物型表型特征,其余251株菌呈现不典型的El Tor生物型表型特征;综合ctxB、rstR基因型和生物型表型特征分析,385 株检测菌株中 64 株为典型 El Tor 生物型菌株,21 株菌有典型的 El Tor 生物型表型特征但携带古典型ctxB基因,杂合型特征菌有280株,根据ctxB、rstR和表型特征的不同组合可再分为45个杂合型。结论 中国O1群El Tor霍乱弧菌菌株呈现明显的表型多态性,传统的表型分型特征不能有效区分古典生物型和El Tor生物型菌株。  相似文献   
7.
8.
ctxB和ureB抗原表位的融合表达及其免疫学活性   总被引:1,自引:0,他引:1  
为研制新型有效的幽门螺杆菌疫苗,设计了一个由ctxB(去除信号肽部分)和ureB抗原表位融合的新型分子。PCR扩增霍乱弧菌的ctxB基因,插入到质粒pEl32a上,再将化学合成的ureB抗原表位序列(编码的氨基酸为CHHLDKSIKEDVQFAQSRI)连接到ctxB下游,融合基因命名为ctube。将含有融合基因的pET32a转化到大肠杆菌BL21(DE3),诱导表达后SDS-PAGE测定融合蛋白ctube的分子质量,ELISA法检测ctube对神经节苷脂GM1的结合活性,Western blotting检测对兔抗幽门螺杆菌(Hp)多抗的结合活性。序列分析结果表明,ctube基因全长387bp,编码129个氨基酸,其中ctxB的密码子与GenBank上的序列同源性达到99%。转化后的大肠杆菌BL21(DE3)经IPTG诱导表达后,产生了一个分子质量为2.5万的蛋白,经肠激酶酶切后的产物为ctube。ELISA试验和West Blotting表明,ctube与神经节苷脂性GM1、兔抗Hp多抗均具有很好的结合活性,有希望成为一个具有抗幽门螺杆菌感染的新的活性分子。  相似文献   
9.
目的构建霍乱孤菌ctxB基因真核表达重组质粒,并在NIH3T3细胞中进行表达。方法用限制性核酸内切酶从重组质粒pET32a-ctxB上切下ctxB基因,导入真核表达载体pcDNA3·1( ),重组子经限制性酶切分析、PCR鉴定正确后,命名为pcDNA3·1-ctxB。用脂质体法将重组质粒pcDNA3·1-ctxB转染NIH3T3细胞,采用免疫荧光法对pcDNA3·1-ctxB的瞬时表达产物进行鉴定。结果约380bp的ctxB被克隆到pcDNA3·1( )真核表达载体中,经测序无误后,用阳离子脂质体转染的方法,检测到重组质粒pcDNA3·1-ctxB在NIH3T3细胞的胞浆和胞膜上得到了表达。结论ctxB真核表达的成功构建及表达为进一步从分子水平研究霍乱肠毒素B亚单位的免疫原性及其作为佐剂的应用价值提供研究基础。  相似文献   
10.
目的初步评价mip基因DNA疫苗的免疫保护性。方法将20只6~8周龄、体重25g左右的BALB/c雌性小鼠。随机分为空白对照组、pcDNA3.1(+)阴性对照组、pcDNA3.1-mip免疫组、pcDNA3.1-mip/ctxB免疫组4个组。每只小鼠股四头肌肌肉注射进行免疫接种,初次免疫2周后加强免疫1次,加强免疫2周后,小鼠腹腔注射嗜肺军团菌L1菌液进行攻击。攻击28d后,检测小鼠肺组织培养带菌量和肺组织病理形态学变化。结果pcDNA3.1-mip免疫组和pcDNA3.1-mip/ctxB免疫组肺组织培养带菌量少于空白对照组和阴性对照组(ANOVA,P〈0.05)。小鼠肺组织肉眼未见明显病理变化,在显微镜下观察肺组织病理切片,发现空白对照组与阴性对照组主要表现为渗出性病变,可见肺泡膈明显增宽,肺毛细血管扩张充血,明显的中性粒细胞浸润,肺泡腔内有渗出物;而应用DNA疫苗的两个免疫组无渗出性病变,pcDNA3.1-mip免疫组肺泡壁和肺间膈轻度增厚,pcDNA3.1-mip/ctxB免疫组肺泡壁和肺泡膈略微增厚。结论应用mip基因DNA疫苗能诱导小鼠在体内产生一定的免疫保护效应。  相似文献   
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