副溶血弧菌TDH的原核表达及单克隆抗体的制备

发布时间：2017-12-28 05:24

本文关键词：副溶血弧菌TDH的原核表达及单克隆抗体的制备　出处：《食品与生物技术学报》2017年03期 　论文类型：期刊论文

【摘要】：为实现副溶血性弧菌tdh基因的原核表达,采用TCBS选择培养基从贝类中筛选副溶血性弧菌疑似菌株;根据Gen Bank上已有的tdh基因序列,设计并人工合成引物,通过PCR技术鉴定副溶血性弧菌并扩增tdh基因;酶切后定向插入到p ET-28a表达载体中,构建重组表达质粒p ET-28a-tdh,转入E.coli Rosetta中,在IPTG诱导下进行TDH蛋白表达。为制备耐热直接溶血毒素TDH单克隆抗体,用纯化的蛋白作为免疫原免疫BALB/c小鼠,成功用原核载体表达的TDH蛋白为免疫原,制得一株能稳定分泌单克隆抗体的杂交瘤细胞,命名为T9N10。获取腹水并经Ni-NTA Resin亲和柱纯化,其稳定分泌的单克隆抗体经鉴定为Ig G1,相对分子质量约为146 000,并表现出较强的特异性。本研究为开发副溶血弧菌免疫学快速检测和深入的研究奠定良好的物质基础。
[Abstract]:In order to realize the prokaryotic expression of TDH gene of Vibrio parahaemolyticus, using TCBS screening of Vibrio parahaemolyticus strains suspected from shellfish culture; according to the TDH gene sequence of Gen Bank on the artificial primers were designed and synthesized by PCR technology, and identification of Vibrio parahaemolyticus and TDH gene amplification; enzyme digestion after inserted into P ET-28a expression vector, the recombinant plasmid P ET-28a-tdh into E.coli, Rosetta, TDH protein expression was induced by IPTG. In order to prepare monoclonal antibodies against heat direct hemolytic toxin TDH, immunized with purified protein, BALB/c mice were successfully immunized with TDH protein expressed in prokaryotic vector. A hybridoma cell secreting monoclonal antibody was named as T9N10. The ascites was purified by Ni-NTA Resin affinity column, and its stable secreted monoclonal antibody was identified as Ig G1, with a relative molecular mass of about 146000, and showed strong specificity. This study provides a good material basis for the development of rapid immunology detection and in-depth study of Vibrio parahaemolyticus.
【作者单位】：浙江医药高等专科学校生物与制药学院;天津市河北区疾病防疫控制中心;浙江大学明州医院;
【基金】：浙江医药高等专科学校教学科研项目(2012024)
【分类号】：R378.3
【正文快照】： Preparation of Monoclonal Antibody against TDH近年来,由感染引起的食物中毒已经成为了严重的食源性公共卫生问题。副溶血性弧菌(VibrioParahaemolyticus,VP)是一种嗜盐性细菌,主要存在于近海岸的鱼类、贝类等海产品中,人食用污染有该致病菌的海产品后可引起胃肠炎,严重时

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