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问号钩端螺旋体lipL32/1-lipL21-OmpL1/2融合基因原核表达及其产物免疫原性分析 被引量:4
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作者 罗冬娇 邱晓枫 +4 位作者 王江 严谨 王海斌 周金成 严杰 《浙江大学学报(医学版)》 CAS CSCD 2008年第6期599-604,共6页
目的:构建问号钩端螺旋体(简称钩体)lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,优化目的产物表达条件并对表达产物免疫原性进行鉴定。方法:采用连接引物PCR构建lipL32/1-lipL21-OmpL1/2融合基因,并用常规基因工程方法建立其原... 目的:构建问号钩端螺旋体(简称钩体)lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,优化目的产物表达条件并对表达产物免疫原性进行鉴定。方法:采用连接引物PCR构建lipL32/1-lipL21-OmpL1/2融合基因,并用常规基因工程方法建立其原核表达系统。采用SDS-PAGE及Bio-Rad凝胶图象分析系统,检测目的重组蛋白rLipL32/1-LipL21-OmpL1/2表达量。采用免疫双扩散试验及WesternBlot,鉴定rLipL32/1-LipL21-OmpL1/2的免疫原性。结果:获得了序列正确的lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统E.coliBL21DE-3pET42a-lipL32/1-lipL21-ompL1/2。表达条件优化后的rLipL32/1-LipL21-OmpL1/2产量为37.78mg/L,是优化前的3.7倍。rLipL32/1-LipL21-OmpL1/2兔抗血清免疫双扩效价为1∶4。rLipL32/1-LipL21-OmpL1/2抗血清能识别rLipL32/1-LipL21-OmpL1/2以及rLipL32/1、rLipL21、rOmpL1/2。rLipL32/1-LipL21-OmpL1/2能与问号钩体56601株全菌兔抗血清以及黄疸出血群、流感伤寒群、波摩那群、秋季群问号钩体感染患者血清出现阳性杂交信号。结论:成功地构建了lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,表达产物具有良好的抗原性和交叉免疫反应性,可作为研制通用型问号钩体基因工程疫苗及通用型钩体病血清学检测的抗原。 展开更多
关键词 钩端螺旋体 问号 抗原 细菌/分析 聚合酶链反应/方法 属特异性抗原 lipL32基 因/lipL21基因/OmpL1/2 融合基因/构建 原核表达 免疫原性/鉴定
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Studies on Pattern of Producing Transgenic Fish II. Several Elements Related to the Establishment of Transgenic Fish
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作者 刘春巧 张永忠 +2 位作者 庄贵洵 孙效文 李云龙 《Developmental and Reproductive Biology》 1997年第2期21-27,共7页
A recombinant plasmid (pcMTsGH) containing the common carp methlothionein promoter (cMT) and salmon growth hormone gene (sGH) was microinjected into common carp fertilized eggs and embryos to optimize the conditions f... A recombinant plasmid (pcMTsGH) containing the common carp methlothionein promoter (cMT) and salmon growth hormone gene (sGH) was microinjected into common carp fertilized eggs and embryos to optimize the conditions for producing transgenic fish. Several key elements were found to have significant effects on the integration of exogenous gene. These elements include forms and doses of pcMTsGH gene microinjected, stages of embryo at which microinjection conducted and pretreatment of fertilized eggs for microinjection. The results show that, in producing transgenic fish, (1) the best time for microinjection is late onecell stage, (2) the most appropriate dose of pcMTsGH gene for microinjection is around 106~107 copies; (3) the hatching rate rises up when the gene is microinjected into the eggs whose chorions are removed, but the integration rate has no significant changes before and after the removal of egg chorions. 展开更多
关键词 transgenic fish integration rate hatching rate
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