单核增生性李氏杆菌溶血素的原核表达及其单克隆抗体的制备

Prokaryotic expression of Listeria monocytogene (LM) hly and development of monoclonal antibodies against listeriolysin O (LLO)

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Western blotting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。

Abstract:

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 1:2×107, 1:2×107 and 1:1×107, 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.