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链霉亲和素-人粒细胞巨噬细胞集落刺激因子融合蛋白的制备与活性鉴定 被引量:1
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作者 孙晶莹 李研 +4 位作者 黄晓燕 靳占奎 徐翠香 常乐 王建华 《陕西医学杂志》 CAS 2023年第1期3-6,22,共5页
目的:制备链霉亲和素(SA)连接的人粒细胞巨噬细胞集落刺激因子(hGM-CSF)融合蛋白(SA-hGM-CSF),并对其生物学活性进行鉴定。方法:在NCBI数据库中查找SA和hGM-CSF序列,将序列合成后连接入Pet28表达载体中,构建SA-hGM-CSF-Pet28重组表达载... 目的:制备链霉亲和素(SA)连接的人粒细胞巨噬细胞集落刺激因子(hGM-CSF)融合蛋白(SA-hGM-CSF),并对其生物学活性进行鉴定。方法:在NCBI数据库中查找SA和hGM-CSF序列,将序列合成后连接入Pet28表达载体中,构建SA-hGM-CSF-Pet28重组表达载体,在BL21(DE3)表达感受态中利用IPTG诱导剂诱导表达,经His镍柱纯化,尿素梯度复性,聚丙烯酰胺凝胶电泳法和蛋白质印迹法(WB)鉴定融合蛋白,通过TF-1细胞检测融合蛋白的生物学活性。结果:SA-hGM-CSF融合蛋白可在BL21(DE3)中高效诱导表达,经His镍柱纯化后该融合蛋白纯度较高。经尿素梯度复性后,细胞学实验验证该融合蛋白具有生物学活性。结论:成功表达具有生物活性的融合蛋白SA-hGM-CSF,该结果为SA-hGM-CSF表面锚定修饰的肿瘤细胞疫苗的研究提供了实验基础。 展开更多
关键词 人粒细胞巨噬细胞集落刺激因子 链霉亲和素 融合蛋白 蛋白纯化 表达载体构建 蛋白活性鉴定
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Identification and characterization of the Vibrio anguillarum prtV gene encoding a new metalloprotease 被引量:3
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作者 莫照兰 郭东升 +4 位作者 茅云翔 叶旭红 邹玉霞 肖鹏 郝斌 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2010年第1期55-61,共7页
We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV inserti... We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In addition,the prtV mutant strain exhibited decreased growth in turbot intestinal mucus and reduced hemolytic activity on turbot erythrocytes.Infection experiments showed that the LD50 of the prtV mutant strain increased by at least 1 log compared to the wild-type in turbot fish.We propose that prtV plays an important role in the pathogenesis of V.anguillarum. 展开更多
关键词 Vibrio anguillarum: prtV PROTEOLYSIS HEMOLYSIS MUCUS LD50
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