[关键词]
[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(Gly4Ser)3将VH基因和VL基因拼接成scFv基因片段,XhoⅠ﹑EcoRⅠ限制性内切酶双酶切scFv后克隆到原核表达载体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-PAGE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。
[Key word]
[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 (Gly4Ser)3 to construct the scFv genetic fragment. The scFv-pET-26b recombinant plasmid was constructed by double-digestion with restriction endonucleases XhoI and EcoRI, followed by cloning into plasmid vector pET-26b. Positive clones were selected for extraction of scFv-pET-26b, which was transformed into Escherichia coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was purified by His affinity chromatography and identified by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). Genetically engineered strains expressing Cronobacter scFv were successfully constructed. SDS-PAGE and ELISA results showed that the molecular weight of the expressed scFv was approximately 30 kDa and that the obtained scFv could specifically bind to Cronobacter and is a potential candidate antibody for the detection of Cronobacter.
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[基金项目]
张家口科技攻关重点资助项目(1311018C-3)