目的 合成并鉴定人参皂苷Re(GRe)人工抗原，用于单克隆抗体的制备。方法 采用高碘酸钠氧化法合成人参皂苷Re人工免疫抗原(GRe-BSA)和人工包被原(GRe-OVA);利用薄层色谱法及紫外光谱法鉴定人参皂苷Re人工免疫抗原和人参皂苷Re人工包被原;利用人参皂苷Re人工免疫抗原多次免疫BALB/c小鼠后，通过间接ELISA法测定免疫小鼠抗血清的效价和特异性。采用PEG法诱导小鼠脾细胞与小鼠骨髓瘤细胞SP2/0融合，有限稀释法进行单克隆细胞株的筛选。结果 紫外光谱和薄层色谱法检测结果表明，人参皂苷Re与BSA/OVA偶联成功;小鼠抗血清效价大于1∶60 000，竞争抑制曲线表明，血清中的抗人参皂苷Re抗体可与人参皂苷Re产生特异性结合。有限稀释法筛选出能分泌抗人参皂苷Re抗体的单克隆细胞株2F4-3H10。结论 成功合成了人参皂苷Re人工免疫抗原和人参皂苷Re人工包被原，并可诱发小鼠产生相应特异性抗体;筛选出了人参皂苷Re单克隆细胞株，可用于腹水的大量制备及免疫组化技术的建立。
Abstract
OBJECTIVE To synthesize and identify a polyclonal or monoclonal antibody against ginseonside Re (GRe). METHODS GRe-BSA and GRe-OVA were synthesized by sodium periodate oxidation method and then examined by UV spectrometry and TLC method. BALB/c mice were immunized with the prepared GRe-BSA conjugate. The titer and specificity of the anti-serum were detected by indirect ELISA and indirect competitive ELISA, respectively. The sprenocytes immunized with GRe-BSA was fused with a mouse myeloma cell line SP2/0. RESULTS According to the UV and TLC, GRe was successfully conjugated to BSA and OVA. The monoclonal antibody against GRe obtained from immunized-mice could bind to GRe specifically and the titer was up to 1∶60 000. A hybridoma secreting monoclonal antibody against ginsenoside Re was screened by limited dilution method.CONCLUSION The artificial GRe antigen is successfully synthesized, which could be used for preparing anti-GRe monoclonal antibody.
关键词
人参皂苷Re /
高碘酸钠氧化法 /
人工抗原 /
单克隆抗体
{{custom_keyword}} /
Key words
ginsenoside Re /
artificial antigen /
synthesis /
monoclonal antibody
{{custom_keyword}} /
中图分类号：
R284
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Ch.P(2005)VolⅡ(中国药典2005年版.二部).2005:62-63.[2] CHEN L M. Studies on effects of ginsenoside on Alzheimer′s disease based on one-compound-multiple-targets strategy. Shenyang: Shenyang Pharmaceutical University,2007.[3] LIU G L. Review of research progress of gisenoside Re. . Jilin Agicultural(吉林农业),2011(3):290.[4] QU H H, ZHANG G L, ZHAO Y. Synthesis and identification of artificial antigen of geniposide . J Beijing Univ Tradit Chin Med (北京中医药大学学报),2013,36(6):387-392.[5] ZHANG Y, QU H H, WU T T. Preparation and identification of monoclonal antibodies against glycyrrhizic acid . Chin J Pharm Anal(药物分析杂志),2013,33(5):770-774.[6] QU H H, ZHAO Y, WANG X Q. Synthesis and identification of baicalin artificial antigen. J Beijing Univ Tradit Chin Med(北京中医药大学学报),2010,33(9):606-609.[7] QU H H, ZHAO Y, WANG Q G. Screening allergens in Chinese medicine injection by imm uno-chip . J Beijing Univ Tradit Chin Med (北京中医药大学学报), 2008, 31(1):23-25.[8] QU H H, ZHAO Y, LI Y F. Key techniques in artificial antigen synthesis of active small molecule in Chinese materia medica . Chin Tradit Herb Drugs(中草药), 2012, 43(10):1880-1885.[9] HONG X Z,SUN M Q. Protein Conjugate Technology (蛋白质连接技术). Beijing: China Medical Science Press,1992:3. LIU Y, QU H H, REN Y. Synthesis and immunogenicity indentification of artificial antigen of ginsenoside Rgl. Chin Tradit Herb Drugs (中草药), 2013, 44(13):1738-1742. SAKATA R, SHOYAMA Y, MURAKAMI H. Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator.Forskolin Cytotechnol, 1994,16(2):101. ZHAO J Z, ZHENG L, XIE W Y. Review of immunoassay of drug residues.Chin Pham J(中国药学杂志),2011,46(2):85-87.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学基金资助项目(81274043,81373542)
{{custom_fund}}
PDF(1043 KB)
