摘要
为制备鼠伤寒沙门菌SseB和SseL蛋白的特异性多克隆抗体(多抗),以鼠伤寒沙门菌S025为供体菌,PCR扩增sseB和sseL基因,插入原核表达载体pGEX-6P-1,转化BL21(DE3)感受态细胞进行诱导表达,获得纯化蛋白免疫小鼠制备多抗;使用λ-red同源重组方法构建sseB和sseL基因缺失株;通过野生株和缺失株的Western-blot分析验证制备血清的特异性。结果显示:成功构建出重组质粒pGEX-6P-1-sseB和pGEX-6P-1-sseL,转化感受态细胞后,在IPTG 1 mmol/L、温度28℃、转速为220 r/min、诱导时间12 h的条件下,SseB和SseL蛋白呈可溶性表达;纯化蛋白免疫小鼠制备了多抗,Western-blot分析显示该多抗可特异性检测鼠伤寒沙门菌中的SseB和SseL蛋白。这两种蛋白多抗的获得为进一步研究其功能奠定基础。
To prepare specific polyclonal antibodies(PcAbs)against proteins SseB and SseL of Salmonella Typhimurium,S.Typhimurium S025 strain was used as the donor strain.sseB and sseL genes were amplified by PCR and inserted prokaryotic expression vector pGEX-6 P-1,the recombinant plasmids were transformed into BL21(DE3)competent cells,respectively.After inducing expression,the purified proteins were used to immunize mice for preparation of PcAbs.The sseB and sseL genes deletion mutants were constructed byλ-red homologous recombination method.The specificity of PcAbs was verified by Western-blot analysis with wild strian and deletion mutants.The results showed that the recombinant plasmids pGEX-6 P-1-sseB and pGEX-6 P-1-sseL were constructed.The soluble fusion proteins of SseB and SseL were obtained when the transformed BL21(DE3)cells were induced by 1 mmol/L IPTG at 28℃for 12 h with rotation of 220 r/min.After purification,the SseB and SseL proteins were immunized mice to prepare PcAbs.The PcAbs could bind specifically to SseB and SseL proteins of wild-type S.Typhimurium by Western-blot analysis.The PcAbs laid a foundation for further study on the function of SseB and SseL proteins.
作者
李海宾
冯政
仲艳
王希玉
李超
秦涛
陈素娟
彭大新
LI Haibin;FENG Zheng;ZHONG Yan;WANG Xiyu;LI Chao;QIN Tao;CHEN Sujuan;PENG Daxin(College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou,Jiangsu 225009;Jiangsu Research Centre of Engineering and Technology for Prevention and Control of Poultry Disease,Yangzhou,Jiangsu 225009)
出处
《中国家禽》
北大核心
2020年第6期42-47,共6页
China Poultry
基金
国家自然科学基金项目(31572530)
江苏省重点研发计划项目(现代农业)(BE2018315)
江苏高校优势学科建设工程。
作者简介
李海宾(1994-),男,硕士研究生,研究方向为预防兽医学,E-mail:79159OO48@qq.com;通讯作者:彭大新(1966-),男,博士,教授,主要从事动物传染病学研究,E-mail:pengdx@yzu.edu.cn。
