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以霍乱毒素B亚基(CTB)基因为载体,构建了含不同抗原表位的恶性疟原虫的融合基因CTB/ATE和CTB/AWTE。前者除含有恶性疟原虫裂殖子表面主要抗原表位杂合多肽基因SPf66外,还含有很强的T辅助细胞表位CST3和Tc细胞表位;后者在此基础上将我国发现的B细胞表位NKNDD基因经8次串联后融合其中。两种形式的融合基因经测序正确后转入大肠杆菌TK1046中,产量分别为10mg/L及5mg/L。表达产物CTB/AWTE经亲和层析纯化双抗夹心ELISA测定表明,该融合蛋白在保留了与抗CTB抗体结合的同时,与抗NKNDD单抗的结合效价达1:8000。 相似文献
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采用遗传工程技术了获得了含有恶性疟原虫子孢子CS抗原融合基因的重组质粒pMC055-CS的工程菌株,能高效分泌CS抗原的融合蛋白至胞外,可达25mg/L,具有霍乱毒素B亚单位和子孢子CS的抗原性;将纯化的融合蛋白免疫C57纯系小鼠,免疫后抗CT-B抗体滴度可达1:3 200-6400,抗CS抗体滴度可达1:320-640,免疫小鼠用纸氏疟孢子腹腔攻击,保护率达34-45%。 相似文献
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将Epstein-Barr病毒(EBV)膜抗原(MA)BLLF1基因,插入含有CMV启动子的真核表达载体pcDNA3下游BamHI位点,构建成真核表达质粒pcDNA3-MA。将纯化的DNA注射Balb/c小鼠股四头肌。经免疫的动物产生抗EB病毒MA特异性的抗体和中和抗体,依赖抗体细胞介导的细胞毒作用(ADCC),特异性T淋巴细胞增生性反应及细胞毒性T淋巴细胞(CTL)杀伤作用。基因免疫与基因-蛋白 相似文献
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Epstein-Barr病毒膜抗原基因免疫的研究 总被引:4,自引:0,他引:4
将Epstein-Bar病毒(EBV)膜抗原(MA)BLLF1基因,插入含有CMV启动子的真核表达载体pcDNA3下游BamHI位点,构建成真核表达质粒pcDNA3-MA。将纯化的DNA注射Balb/c小鼠股四头肌。经免疫的动物产生抗EB病毒MA特异性的抗体和中和抗体,依赖抗体细胞介导的细胞毒作用(ADCC),特异性T淋巴细胞增生性反应及细胞毒性T淋巴细胞(CTL)杀伤作用。基因免疫与基因-蛋白联合免疫效果相似。不同启动子控制下的MA基因,诱发小鼠免疫应答无明显差异。 相似文献
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采用遗传工程技术获得了含有恶性疟原虫子孢子CS抗原融合基因的重组质粒pMC055-CS的工程菌株,能高效分泌CS抗原的融合蛋白至胞外,可达25mg/L.具有霍乱毒素B亚单位(CT-B)和子孢子CS的抗原性.将纯化的融合蛋白免疫C57纯系小鼠,免疫后抗CT-B抗体滴度可达1:3200~6400,抗CS抗体滴度可达1:320~640.免疫小鼠采用约氏疟子孢子腹腔攻击,保护率达34~45%. 相似文献
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合成了5对寡核苷酸片段,分别连接在两种恶性疟原虫杂合多肽(45肽和58肽)抗原基因片段(HPFGA和HPFGB)的头部和尾部,将这两种片段分别与霍乱毒素B亚单位(CT-B)基因末端融合。两种杂合多肽抗原分别含有数个红内期和红外期有代表性的、并能被T或B淋巴细胞识别的保护性抗原表位,CT-B基因前端具有促使分泌的信号肽序列。将这两种融合基因的不同重组质粒分别转化大肠杆菌,对转化菌的培养上清检测表明融合蛋白被分泌性表达,既具有恶性疟原虫和CT-B抗原性,又保持了CT-B与其受体神经节苷脂CM1结合的生物学活性。 相似文献
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恶性疟原虫多抗原表位基因表达载体的构建及其在大豆幼胚中的表达 总被引:2,自引:0,他引:2
疟疾是当今最需要研究有效疫苗的主要传染病之一。AWTE基因编码恶性疟原虫多种抗原表位基因 ,CTB基因编码霍乱毒素 B亚基 ,是一种既能引起细胞免疫又能引起体液免疫的免疫载体和佐剂。把 AWTE- CTB融合基因构建到植物表达载体 p BVG- ny2上 ,通过基因枪导入法 ,转化大豆幼胚分生组织。 X- glu染色检测到 GUS基因的表达 ;抗原性分析实验结果表明 ,特异表达的融合蛋白可与 CTB和 AWTE抗体结合 ,具有 CTB抗原性。这个实验结果 ,表明疟疾多抗原表位基因首次在转基因大豆幼胚中得到瞬时表达 相似文献
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Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule 总被引:5,自引:0,他引:5
J R Oksenberg C Ko A K Judd M Lim A Kent G K Schoolnik L Steinman 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(12):4227-4231
The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT. 相似文献
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James Terrell Preeti Yadava Carlos Castro Jeffrey Hughes 《Journal of liposome research》2013,23(1):21-29
Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins. 相似文献
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Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins. 相似文献
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Possible involvement of a GTP-binding protein in a late event during endogenous ganglioside-modulated cellular proliferation 总被引:3,自引:0,他引:3
S Spiegel 《The Journal of biological chemistry》1989,264(12):6766-6772
The B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts as measured by an increase in [3H]thymidine incorporation. Pertussis toxin pretreatment markedly inhibits B subunit-induced DNA synthesis. The inhibitory effects of pertussis toxin were observed even in the presence of insulin which greatly potentiates the mitogenic response to the B subunit. Treatment with either pertussis toxin or insulin did not alter the binding of the B subunit to the cells. The dose-response for pertussis toxin-induced inhibition of DNA synthesis correlated closely with the dose-response for ADP-ribosylation of a 41-kDa membrane protein, suggesting the involvement of a GTP-binding protein that is a substrate for pertussis toxin (Gi) in mitogenesis induced via cross-linking of endogenous gangliosides. Pertussis toxin, in a similar concentration-dependent manner, also inhibited the mitogenic response to unfractionated fetal calf serum and to bombesin in the absence or presence of insulin. The inhibitory effect of pertussis toxin was clearly unrelated to any effects on known G proteins coupled to adenylate cyclase or phospholipase C. In addition, pertussis toxin did not impair the early increase in cytosolic free Ca2+ induced by the B subunit or bombesin. Pertussis toxin-induced inhibition of DNA synthesis could still be observed even when the toxin was added as late as 6 h after addition of the growth-promoting agents. This suggests the involvement of a GTP-binding protein in a late step of the B subunit- and bombesin-mediated pathways of mitogenesis. The possibility that other growth factors bypass this pathway is shown by their lack of sensitivity to pertussis toxin. 相似文献
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The bimodal growth response of Swiss 3T3 cells to the B subunit of cholera toxin is independent of the density of its receptor, ganglioside GM1 总被引:2,自引:0,他引:2
The B subunit of cholera toxin, a protein which binds specifically to cell surface ganglioside GM1, has been shown to have a bimodal effect on DNA synthesis in Swiss 3T3 fibroblasts. The B subunit induced cellular proliferation of confluent and quiescent cells while it inhibited the growth of the same cells when they were sparse and rapidly dividing. The amount of cell surface GM1 increased when the cells reached confluency. To examine the hypothesis that the variation in levels of GM1 was responsible for the bimodal effect, we increased GM1 levels in rapidly dividing cells by insertion of exogenous GM1 or by treatment of the cells with neuraminidase to convert polysialogangliosides to GM1. Even after the level of GM1 was increased to levels similar to those found in confluent cells, the B subunit still inhibited, rather than stimulated, their growth. Therefore, this result indicates that the bimodal response to the B subunit is not solely a function of the concentration of cell surface GM1; rather it is the growth stage that determines the fate of the signal transduced by the interaction of the B subunit and ganglioside GM1. 相似文献
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T Tetsumoto K Takada N Amino K Miyai 《Biochemical and biophysical research communications》1988,157(2):605-610
In quiescent rat thyroid (FRTL-5) cells, the B subunit of cholera toxin, which binds to cell surface ganglioside GM1 specifically, alone induced DNA synthesis and markedly enhanced that induced by insulin in serum-free medium. On the other hand, the B subunit inhibited DNA synthesis induced by thyrotropin (TSH). The B subunit did not activate adenylate cyclase and had no effect on the TSH-induced cyclic adenosine 3',5'-monophosphate (cAMP) production. Moreover, the B subunit inhibited DNA synthesis induced by dibutyryl cAMP (Bt2cAMP) or phorbol-12-myristate-13-acetate (PMA). These data demonstrate that the B subunit has both stimulatory and inhibitory effects on DNA synthesis in FRTL-5 cells depending on the presence of other growth factors and that these effects on cell proliferation by the interaction of the B subunit, possibly with cell surface ganglioside GM1, may involve a mechanism independent from the modulation of membrane receptor function through interaction with growth factor receptor. 相似文献