肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定

doi:10.3969/j.issn.1004-616x.2014.03.013

癌变·畸变·突变

肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定

宋巍，杨海波，陈兰英*

河南城建学院生命科学与工程学院，河南平顶山 467044

收稿日期:2013-09-04 修回日期:2014-01-24 出版日期:2014-05-30 发布日期:2014-05-30
通讯作者: 陈兰英，E-mail：lychen666@163.com
作者简介:宋巍(1982- )，男，博士，讲师，研究方向：肿瘤细胞生物学。
基金资助:
河南省重点科技攻关项目(092102310006)，河南城建学院校内基金(2010JYB009)

Preparation and identification of monoclonal antibody against liver fatty acid binding protein

SONG Wei，YANG Hai-bo，CHEN Lan-ying*

College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan 467044, Henan, China

Received:2013-09-04 Revised:2014-01-24 Online:2014-05-30 Published:2014-05-30

摘要/Abstract

摘要：

目的：制备抗肝脏型脂肪酸结合蛋白(LFABP)的单克隆抗体(mAb)并进行亚型和特异性鉴定。方法：以重组的LFABP蛋白为免疫原，经过原核表达和纯化后，免疫BALB/c小鼠，获得分泌鼠抗人LFABP蛋白mAb的杂交瘤细胞株，通过ELISA 和Western blot方法检测其特异性。结果：纯化的LFABP重组蛋白免疫小鼠后经过筛选得到2株稳定分泌抗人LFABP的mAb杂交瘤细胞株，分别命名为3E6和5B7，其亚型分别为IgG1和IgG2a，抗体经纯化后浓度达到2 mg/mL，效价达到1∶10 000以上。Western blot分析结果表明可与细胞中表达的内源LFABP发生特异性免疫反应。结论：成功制备了鼠抗人LFABP的mAb，为进一步研究LFABP的生物学特性，并为相关疾病的治疗奠定了基础。

关键词: 肝脏型脂肪酸结合蛋白, 原核表达, 杂交瘤, 单克隆抗体

Abstract:

OBJECTIVE: To prepare anti-liver-type fatty acid binding protein (LFABP) monoclonal antibody (mAb) and identified subtype and specificity. METHODS：The LFABP recombinant protein was used to immunize BALB/c mice，spleen cells from immunized mice were fused with myeloma cell Sp2/0 . After HAT selective culture and indirect ELISA screening，we obtained hybridoma cell line secreting mouse mAb against human LFABP. The specificity was verified by ELISA and Western blotting. RESULTS：After using purified recombinant protein immunized mice and screening，we obtained two hybridoma cell lines secreting the mAb against human LFABP，named 3E6 and 5B7，both were of the IgG1 subtype. The antibody was purified，reaching a concentration of 2 mg/mL，and titer of 1∶10 000 above. Western blotting showed that mAb had a specific reaction with the LFABP endogenous expression in the cells. CONCLUSION：We successfully prepared mice mAb against human LFABP ，which could not only provide an important foundation to further studies of the biological characteristics of LFABP，mechanism involved in regulation of fatty acid metabolism and drug interaction，but also provide experimental evidence for the treatment of related diseases.

Key words: liver-type fatty acid binding protein, prokaryotic expression, hybridoma, monoclonal antibody

宋巍，杨海波，陈兰英*. 肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2014.03.013.

SONG Wei，YANG Hai-bo，CHEN Lan-ying*. Preparation and identification of monoclonal antibody against liver fatty acid binding protein[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2014.03.013.

参考文献

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肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibody against liver fatty acid binding protein

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