基于ELISA克罗诺杆菌单链抗体制备与鉴定.pdf

现代食品科技 Modern Food Science and Technology 2015, Vol.31, No.5 基于ELISA 克罗诺杆菌单链抗体制备与鉴定 张秀媛，黄智鸿，王丽霞，陈一，赵瑞平 (河北北方学院农林科技学院，河北北方学院食品安全研究中心,河北张家口 075000) 摘要：利用基因工程技术制备克罗诺杆菌特异性单链抗体（scFv ）。从克罗诺杆菌单克隆抗体的杂交瘤细胞中提取总 RNA，利 用RT-PCR 反转录合成第一链cDNA 后再扩增出抗体的重链可变区(VH)和轻链变区(VL)基因片段，采用重叠延伸PCR 的方法，用柔 性多肽 Linker 接头（Gly Ser) 将VH 基因和VL 基因拼接成scFv 基因片段，Xho Ⅰ﹑EcoR Ⅰ限制性内切酶双酶切scFv 后克隆到原核 4 3 表达载体pET-26b 中构建重组质粒scFv-pet -26b ，挑取阳性克隆提取重组质粒后转入大肠杆菌BL21 中进行诱导表达，通过His 柱进 行亲和层析，最后利用ELISA 检测单链抗体的活性。成功构建了表达克罗诺杆菌单链抗体的基因工程菌株，通过 SDS和ELISA 试验结果表明，诱导表达的罗诺杆菌单链抗体分子量约为30 kDa，其能与罗诺杆菌特异性结合，可作为免疫检测罗诺杆菌的候选抗 体分子。 关键词：克罗诺杆菌；scFv ；ELISA 文章篇号：1673-9078(2015)5-169-174 DOI: 10.13982/j.mfst.1673-9078.2015.5.027 Preparation and Identification of Cronobacter Single-chain Variable Fragment Antibody using ELISA ZHANG Xiu-yuan, HUANG Zhi-hong, WANG Li-xia, CHEN Yi, ZHAO Rui-ping (College of Agriculture and Forestry Science and Technology, Hebei North University; Food Safety Research Centre of Hebei North University, Zhangjiakou 075000, China) Abstract: Specific single-chain variable fragment (scFv) against Cronobacter was generated using genetic engineering. Genes coding for variable regions of the heavy and light chains (VH and VL) were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA from hybridoma cells secreting Cronobacter monoclonal antibody. VH and VL were fused together by Splice Overlap Extension (SOE)-PCR with flexible polypeptide linker (Gly Ser) to construct the scFv genetic fragment. The scFv-pET-26b recombinant plasmid was 4 3 constructed by double-digestion with restriction endonucleases