霍乱毒素的纯化及其单克隆抗体的制备与初步应用
发布时间：2019-06-17 13:25
【摘要】： 目的建立从培养基上清分离纯化霍乱毒素的有效方法,制备高纯度的霍乱毒素;获得稳定分泌抗霍乱毒素的杂交瘤细胞株、纯化抗霍乱毒素的单克隆抗体和多克隆抗体;克隆大肠不耐热肠毒素基因、构建其表达载体、纯化并鉴定大肠不耐热肠毒素,以对抗霍乱毒素单克隆抗体的特异性进行评价;用生物素标记抗霍乱毒素单克隆抗体、筛选最佳的夹心ELISA抗体组合、确定抗体最佳包被浓度、建立生物素—亲和素—ELISA的方法检测CT,以期为霍乱弧菌产毒株的诊断奠定基础。 方法将霍乱弧菌O1群569B在AKI培养基中培养,收集上清,用六偏聚磷酸钠盐析,磷酸纤维素P11柱和Immobilized D-galactose柱纯化,超滤浓缩后获得CT,SDS-PAGE、Western Blot和GM1-ELISA验证其纯度和生物学活性。以纯化的CT免疫Balb/c小鼠,采用传统的杂交瘤技术制备针对CT的单克隆抗体(MAb),以间接法ELISA法筛选稳定分泌抗CT的杂交瘤细胞株,体内法诱导产生腹水,鉴定MAb类型,采用优球蛋白法纯化IgM抗体,HiTrap rProtein A FF柱纯化多克隆抗体。PCR扩增大肠杆菌E10407的LT基因,酶切后连接于pUC-19质粒,设计引物进行点突变,然后连接于表达载体pET-30a,测序验证序列的正确,并转化于大肠杆菌BL21(DE3)。GM1-ELISA筛选表达LT的阳性克隆,超声破碎重组大肠菌,用Immobilized D-galactose柱纯化LT蛋白,SDS-PAGE、质谱和Vero细胞毒性试验验证其纯度及生物学活性。用生物素标记抗霍乱毒素单克隆抗体,筛选最佳的夹心ELISA抗体组合,确定最适的抗体包被浓度和一抗稀释倍数,建立BA-ELISA法检测CT。确定最低检测CT的阈值,并用BA-ELISA法检测不同细菌的培养基上清。 结果获得相对分子量约为85kDa的高纯度且有生物学活性的CT蛋白和5株稳定分泌抗CT MAb的杂交瘤细胞株,抗体类型均为IgM,经ELISA证实其可结合CT,其中两株结合于CT-B亚单位,其余三株的既可结合于CT-A,也可结合于CT-B。获得了高纯度的抗霍乱毒素单克隆抗体和多克隆抗体。制备了高纯度且有生物学活性的LT蛋白。确定了以抗CT多抗(CTPAb)为包被抗体,生物素标记的抗CT-B IgG单抗(CTMAb-XU-22)为一抗的夹心BA-ELISA的最佳检测组合,抗CT多抗的最佳包被浓度为20μg/ml,抗CT-B IgG单抗的最佳稀释倍数为1:50,最低检测CT的限值为2ng。BA-ELISA法检测不同细菌培养液的上清的结果与PCR法一致,并可区分霍乱弧菌的产毒株和非产毒株。 结论本研究建立了从霍乱弧菌培养基上清分离纯化CT蛋白的有效方法,获得了高纯度和高活性的CT和LT蛋白,制备了抗CT MAb和PAb,利用多克隆抗体的强富集能力、单克隆抗体的高度特异性以及生物素—亲和素系统的放大作用,建立了高灵敏度的生物素—亲和素—ELISA的方法,为进一步应用此检测方法快速检测霍乱弧菌,对霍乱弧菌产毒株,特别是毒素基因转移的新致病菌株的发现、监测、预防和控制工作有很大的帮助。
[Abstract]:Objective to establish an effective method for isolation and purification of cholera toxin from culture medium, to prepare high purity cholera toxin, to obtain hybridoma cell line stably secreting anti-cholera toxin, to purify monoclonal antibody and polyclonal antibody against cholera toxin, to clone colorectal heat-resistant enterotoxin gene, to construct its expression vector, to purify and identify colorectal heat-resistant enterotoxin, and to evaluate the specificity of monoclonal antibody against cholera toxin. The monoclonal antibody against cholera toxin was labeled with biotin, the best combination of sandwich ELISA antibody was screened, the optimal coating concentration of antibody was determined, and the method of biotin-avidin-ELISA was established to detect CT, in order to lay a foundation for the diagnosis of Vibrio cholera strain. Methods Vibrio cholera O1 group 569B was cultured in AKI medium and purified by sodium hexametaphosphate salting out, cellulose phosphate P11 column and Immobilized D-galactose column. after ultrafiltration concentration, CT,SDS-PAGE,Western Blot and GM1-ELISA were obtained to verify its purity and biological activity. Balb/c mice were immunized with purified CT. Monoclonal antibody (MAb), against CT was prepared by traditional hybridoma technique. Hybridoma cell line stably secreting anti-CT was screened by indirect ELISA method. Ascitic fluid was induced by in vivo method, MAb type was identified, IgM antibody, HiTrap rProtein A FF column was purified by euglobulin method. LT gene of E. coli E10407 was amplified by PCR and ligated to pUC-19 plasmid. Primers were designed for point mutation, then ligated to the expression vector pET-30a, sequencing to verify the correct sequence, and transformed into E. coli BL21 (DE3). The positive clones expressing LT were screened by GM1-ELISA, the recombinant coliform bacteria were broken by ultrasound, and the LT protein was purified by Immobilized D-galactose column. the purity and biological activity of LT protein were verified by SDS-PAGE, mass spectrometry and Vero cytotoxicity test. The monoclonal antibody against cholera toxin was labeled with biotin, the best sandwich ELISA antibody combination was screened, the optimum antibody coating concentration and dilution multiple were determined, and the BA-ELISA method was established for the detection of CT.. The minimum threshold for detection of CT was determined, and the culture medium of different bacteria was detected by BA-ELISA. Results High purity and biologically active CT protein with relative molecular weight of 85kDa and 5 hybridoma cell lines stably secreting anti-CT MAb were obtained. The antibody types of IgM, were confirmed by ELISA to bind to CT, two of which could bind to CT-B subunit, and the other three strains could bind to both CT-A, and CT-B.. High purity monoclonal antibodies and polyclonal antibodies against cholera toxin were obtained. LT protein with high purity and biological activity was prepared. The optimum detection combination of sandwich BA-ELISA with anti-CT polyclonal antibody (CTPAb) as coating antibody and biotin-labeled anti-CT-B IgG monoclonal antibody (CTMAb-XU-22) as primary antibody was determined. the optimum coating concentration of anti-CT polyantibody was 20 渭 g / ml, anti-CT-B IgG monoclonal antibody was 1 鈮，
本文编号：2501020
[Abstract]:Objective to establish an effective method for isolation and purification of cholera toxin from culture medium, to prepare high purity cholera toxin, to obtain hybridoma cell line stably secreting anti-cholera toxin, to purify monoclonal antibody and polyclonal antibody against cholera toxin, to clone colorectal heat-resistant enterotoxin gene, to construct its expression vector, to purify and identify colorectal heat-resistant enterotoxin, and to evaluate the specificity of monoclonal antibody against cholera toxin. The monoclonal antibody against cholera toxin was labeled with biotin, the best combination of sandwich ELISA antibody was screened, the optimal coating concentration of antibody was determined, and the method of biotin-avidin-ELISA was established to detect CT, in order to lay a foundation for the diagnosis of Vibrio cholera strain. Methods Vibrio cholera O1 group 569B was cultured in AKI medium and purified by sodium hexametaphosphate salting out, cellulose phosphate P11 column and Immobilized D-galactose column. after ultrafiltration concentration, CT,SDS-PAGE,Western Blot and GM1-ELISA were obtained to verify its purity and biological activity. Balb/c mice were immunized with purified CT. Monoclonal antibody (MAb), against CT was prepared by traditional hybridoma technique. Hybridoma cell line stably secreting anti-CT was screened by indirect ELISA method. Ascitic fluid was induced by in vivo method, MAb type was identified, IgM antibody, HiTrap rProtein A FF column was purified by euglobulin method. LT gene of E. coli E10407 was amplified by PCR and ligated to pUC-19 plasmid. Primers were designed for point mutation, then ligated to the expression vector pET-30a, sequencing to verify the correct sequence, and transformed into E. coli BL21 (DE3). The positive clones expressing LT were screened by GM1-ELISA, the recombinant coliform bacteria were broken by ultrasound, and the LT protein was purified by Immobilized D-galactose column. the purity and biological activity of LT protein were verified by SDS-PAGE, mass spectrometry and Vero cytotoxicity test. The monoclonal antibody against cholera toxin was labeled with biotin, the best sandwich ELISA antibody combination was screened, the optimum antibody coating concentration and dilution multiple were determined, and the BA-ELISA method was established for the detection of CT.. The minimum threshold for detection of CT was determined, and the culture medium of different bacteria was detected by BA-ELISA. Results High purity and biologically active CT protein with relative molecular weight of 85kDa and 5 hybridoma cell lines stably secreting anti-CT MAb were obtained. The antibody types of IgM, were confirmed by ELISA to bind to CT, two of which could bind to CT-B subunit, and the other three strains could bind to both CT-A, and CT-B.. High purity monoclonal antibodies and polyclonal antibodies against cholera toxin were obtained. LT protein with high purity and biological activity was prepared. The optimum detection combination of sandwich BA-ELISA with anti-CT polyclonal antibody (CTPAb) as coating antibody and biotin-labeled anti-CT-B IgG monoclonal antibody (CTMAb-XU-22) as primary antibody was determined. the optimum coating concentration of anti-CT polyantibody was 20 渭 g / ml, anti-CT-B IgG monoclonal antibody was 1 鈮，
本文编号：2501020
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