摘要
为了深入研究单核增生性李氏杆菌(LM)致病机理,从其基因组中克隆李氏杆菌溶血素基因hly,并将其与原核表达载体连接在大肠杆菌BL21中表达携带His标签的李氏杆菌溶血素(LLO)融合蛋白,经镍柱纯化得到重组LLO蛋白作为免疫原并免疫小鼠。取免疫小鼠的脾细胞与骨髓瘤细胞(Sp2/0)进行融合,经过3次亚克隆后获得3株稳定分泌针对LLO蛋白单抗的杂交瘤细胞株,分别命名为Anti-LLO1、Anti-LLO2、Anti-LLO3;经ELISA测定其细胞培养上清效价分别为1:3.6×104、1:6.4×104、1:1.6×104,腹水效价分别为1:2×107、1:2×107、1:1×107;亲和力解离常数(Kd)分别为6.18×10-11、7.50×10-11、6.27×10-11;3株单抗的IgG亚类均为IgG1。经Westernblotting鉴定证明,该3株抗体均能特异地识别李氏杆菌LLO蛋白,该单抗的制备为深入研究LM的致病机理奠定了基础。
In order to study the pathogenesis of Listeria monocytogenes（LM）,we cloned listeriolysin gene into prokaryotic expression vector PET21a.The expression vector was transformed into Escherichia coli BL21 for expression of listeriolysin O（LLO）.LLO-His tag fusion protein was purified with a Ni-NTA affinity column and was used as an immunogen to vaccinate BALB/C mice.Hybridomas were developed by fusing mouse myeloma cells Sp2/0 and splenocytes from the immunized mice and screened with purified LLO.Three hybridomas secreting antibodies against listeriolysin O were obtained and named anti-LLO1,anti-LLO2 and anti-LLO3,respectively.Western blotting analysis showed that all of them could specifically bind to the LLO secreted by the LM.The titers of anti-LLO monoclonal antibodies in the supernatants of three hybridomas cultures were 1：3.6×104,1：6.4×104 and 1：1.6×104,respectively,and the titers of ascites from the hybridoma-injected mice were 1：2×10^7,1：2×10^7 and 1：1×10^7,respectively,based on ELISA test.The isotypes of the monoclonal antibodies were determined to be IgG1.The dissociation constants（Kd） of these three monoclonal antibodies were determined to be 6.18×10^-11,7.50×10^-11 and 6.27×10^-11 respectively.These data and reagents will be of great assistance to elucidate the pathogenesis of Listeriosis.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第11期1652-1657,共6页
Chinese Journal of Biotechnology
基金
国家重点基础研究发展计划(973计划)(No.2006CB504303)
国家自然科学基金项目(No.30725026)资助~~
