摘要
目的表达高纯度的抗苗勒管激素蛋白,以该蛋白作为抗原制备单克隆抗体,并鉴定抗体亚型,为进一步开发优质的抗苗勒管激素诊断试剂盒做技术储备。方法在表达载体pET⁃28a(+)质粒中接入经密码子优化的抗苗勒管激素基因(AMH)片段,转化于BL21细菌中,异丙基硫代⁃β⁃D⁃半乳糖苷(IPTG)诱导重组蛋白大量表达,对上清中可溶性蛋白进行纯化。使用SDS⁃PAGE分析纯化蛋白的纯度。经纯化的抗原蛋白3次皮下免疫小鼠,挑选产生高效价的抗体的小鼠,取其脾细胞,制备悬液。经细胞杂交技术与骨髓瘤细胞进行融合,筛选能稳定持久分泌抗AMH抗体的杂交瘤细胞株,并制备腹水抗体。通过亲和层析技术、BCA蛋白检测方法等测定手段,纯化抗体、检测抗体浓度。结果筛选到2株能持久分泌抗AMH蛋白单克隆抗体的杂交瘤细胞株,分别为克隆细胞株3F6和4E7,分泌抗体均为IgG1亚型。结论筛选出稳定分泌高纯度的抗苗勒管激素抗体的2株杂交瘤细胞,并制备单克隆抗体。下一步将其应用于AMH检测试剂盒的开发。
Objective To express a high⁃purity anti⁃Mullerian hormone protein antigen,and use this antigen to prepare a monoclonal antibody,to identify antibody subtypes,and to provide antigen and antibody materials for the development of a high⁃quality anti⁃Mullerian hormone diagnostic kit.Methods Following E.coli expression system codon protocols,a codon⁃optimized anti⁃Mullerian hormone(AMH)gene fragment was subcloned into the expression vector pET⁃28a(+)and transformed into E.coli BL21.The AMH amino acid sequence was conserved.Before and after the optimization process,the prokaryotic expression vector and host bacteria were examined for protein expression.The recombinant AMH protein was induced,expressed and purified by His⁃tag purification.The purified recombinant protein activity was confirmed by western blot analysis.BALB/c mice were immunized subcutaneously three times with the purified protein and serum anti⁃AMH antibody titers were measured by ELISA.When the titer reached 1∶80,000,the mouse spleen cells were collected to prepare a suspension and fused with BALB/c mouse myeloma cells.The hybridoma cell lines stably and persistently secreting anti⁃AMH antibodies were screened and identified.The anti⁃AMH monoclonal antibody was purified and validated.Results Two hybridoma cell lines,3F6 and 4E7,were established and produced anti⁃AMH specific monoclonal antibodies.Both 3F6 and 4E7 monoclonal antibodies were IgG1 subtypes.Conclusion Two anti⁃AMH specific monoclonal antibodies,3F7 and 4E6,were identified and validated.The next step is to apply these monoclonal antibodies to develop AMH detection kits,promoting the development of domestic diagnostic reagents.
作者
苏晓菱
李丽蓥
李苗苗
何洁
王瑞雪
王勇
罗树红
SU Xiaoling;LI Liying;LI Miaomiao;HE Jie;WANG Ruixue;WANG Yong;LUO Shuhong(Department of Laboratory Medicine,School of Stomatology and Medicine,Foshan University,Foshan,Guangdong,China,528000;Guangzhou Hongxiang Biological Medicine Technology Co.,Ltd.,Guangzhou,Guangdong,China,510663)
出处
《分子诊断与治疗杂志》
2020年第2期239-243,共5页
Journal of Molecular Diagnostics and Therapy
基金
佛山科学技术学院大学生创新创业训练项目(201811847075)
佛山大学高层次人才启动资金(20161110004)
佛山大学高水平大学建设基金(20170131020)。
关键词
抗苗勒管激素
单克隆抗体制备
蛋白纯化
Anti-Mullerian hormone
Monoclonal antibody
Hyridoma
Protein expression
