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目的:利用基因工程技术制备抗肠炎沙门氏菌的单链抗体。方法:从抗肠炎沙门氏菌单克隆抗体的杂交瘤细胞中纯化RNA,反转录后扩增出抗体的重链可变区(VH)和轻链可变区(VL)基因片段,采用重叠延伸的方法,用柔性多肽Linker接头(Gly4Ser)3按VL-Linker-VH方式将VH基因和VL基因拼接成单链抗体基因片段后,连接到pGEX-4T-1载体上,进行重组转化。挑取阳性克隆,经IPTG诱导后,通过GST柱进行亲和层析,最后利用ELISA检测抗体的活性。结果:成功构建了表达抗肠炎沙门氏菌单链抗体的基因工程菌株,经SDS-PAGE和ELISA检测结果表明,诱导表达的单链抗体scFv分子量约为60 kDa,其能特异与肠炎沙门氏菌结合,但与副甲伤寒沙门氏菌、鸭沙门氏菌、鼠伤寒沙门氏菌有轻度交叉反应。结论:成功构建了抗肠炎沙门氏菌单链抗体的表达菌株,表达的单链抗体scFv可作为沙门氏菌的检测的候选抗体分子。
OBJECTIVE: To prepare single chain antibodies against Salmonella Enteritidis using genetic engineering techniques. METHODS: RNA was purified from hybridomas of anti-Salmonella enteritidis monoclonal antibody and reverse-transcribed to amplify the variable region (VH) and variable region (VL) genes of the antibody. Methods: The VH gene and the VL gene were spliced into the single-chain antibody gene fragment by the VL-Linker-VH method using the flexible polypeptide Linker linker (Gly4Ser) 3, and then ligated into pGEX-4T-1 vector for recombinant transformation. Positive clones were picked, induced by IPTG, and then subjected to affinity chromatography by GST column. Finally, the activity of the antibody was detected by ELISA. Results: The recombinant strain of Salmonella enteritidis scFv was constructed successfully. SDS-PAGE and ELISA showed that the molecular weight of the scFv was about 60 kDa, which could specifically bind Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium have a mild cross-reactivity. CONCLUSION: The constructed S. sclerotiorum single chain antibody was successfully constructed. The expressed scFv antibody could be used as a candidate antibody for detection of Salmonella.