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2025年8月29日 星期五
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抗人红细胞单链抗体与猪伪狂犬gE蛋白双功能融合蛋白的构建及生物学活性检测
廖文军
广西兽医研究所
Construction and Biologic Activity Detection of Anti-Human Red Blood Cell ScFv-PRV gE Bifunctional Fusion Protein
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摘要 构建既具有凝集性又具有抗原性的双功能融合蛋白,本研究利用特异性引物从重组质粒PMD-2E8ScFv和PMD-gE中PCR扩增得2E8基因(抗人红细胞H抗原单链抗体基因)和kgE基因(PRVgE主要抗原编码区),采用重叠延伸(SOE)PCR法拼接成融合的双功能分子 2E8kgE,经BamHI和EcoRI双酶切处理,纯化后,克隆至BamHI和EcoRI双酶切处理的表达载体pET-Trx中,构建了原核表达载体pET-Trx-2E8kgE;将pET-Trx-2E8kgE转化至BL21plysS中,在IPTG的诱导下表达具有双功能的融合蛋白,经过SDS-PAGE和Western-blot检测;结果表明,重组菌BL21plysS表达出约60.1kDa的目的蛋白,与猪伪狂犬标准阳性血清发生特异性反应。红细胞凝集试验证实:2E8kgE融合蛋白既能够与人红细胞结合,又能够与PRV抗体反应,具有双功能特性。
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廖文军
关键词 关键词:猪伪狂犬病毒 gE基因 原核表达    
Abstract:Construction of bifunctional fusion protein which can conjugate both human red blood cell and antibody against PRV.2E8 gene(ScFv gene against H antigen of human erythrocytes)and kgE gene (the major epitope domain of glycoprotein gE of pseudorabies virus) ,amplified from the recombinant plasmid PMD-2E8ScFv and PMD-gE,respectvely,were spliced by overlaped extension PCR and the assembled bifunctional molecule was named 2E8kgE.The 2E8kgE digested with BamHI and EcoRI was inserted into pET-Trx vector,to yield the recombinan vector pET-Trx-2E8kgE.Then the recombinan vector pET-Trx-2E8kgE was transformed into BL21plysS and protein expressions was induced with IPTG.SDS-PAGE and western-blot analyses showed that the fusion peotein was 60.1kDa and the protein was specific to antisera against PRV. Erythrocyte agglutination test results indicated that 2E8kgE fusion protein can conjugate both human red blood cell and antibody against PRV.
Key wordsKey words:PRV    gE gene    prokaryotic expression
收稿日期: 2009-05-04     
通讯作者: 廖文军   
引用本文:   
廖文军. 抗人红细胞单链抗体与猪伪狂犬gE蛋白双功能融合蛋白的构建及生物学活性检测[J]. , 2010, 18(3): 562-566.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2010/V18/I3/562
 
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