抗人AEG-1单克隆抗体可变区基因克隆及序列分析

doi:10.13523/j.cb.20141202

中国生物工程杂志

2014, Vol. 34

Issue (12): 10-15 DOI: 10.13523/j.cb.20141202

研究报告

抗人AEG-1单克隆抗体可变区基因克隆及序列分析

龙敏, 董柯, 王希, 陈曦, 刘冲, 刘丽, 张惠中

第四军医大学唐都医院西安 710038

Developing an Anti-human AEG-1 Monoclonal Antibody Secreting Hybridoma and Its Variable Region Fragment Amplification

LONG Min, DONG Ke, WANG Xi, CHEN Xi, LIU Chong, LIU Li, ZHANG Hui-zhong

Department of Clinical Research and Diagnosis, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China

全文: PDF(792 KB) HTML

摘要：

AEG-1基因位于人染色体8q22,编码582个氨基酸,参与多种信号转导途径并与多种恶性肿瘤的发生、发展及生物学表型密切相关。为更好地探讨AEG-1生物学功能,以纯化的pGSTag-AEG-1蛋白免疫BALB/c小鼠,应用细胞融合技术并经筛选及鉴定,获得了分泌抗人AEG-1单克隆抗体的杂交瘤细胞株1E3;Western blot及免疫组化证实该细胞株分泌的单克隆抗体能与肿瘤细胞中AEG-1蛋白特异性结合;RT-PCR方法从1E3细胞中克隆出抗AEG-1抗体的VH和VL基因片段,通过测序分析、碱基和蛋白序列的比对确认该株抗体为鼠源性IgG的轻、重链可变区基因。进一步运用Kabat System在线分析系统对VH和VL基因进行结构分析,确证FWRs和CDRs的结构完整,VH编码117个氨基酸;VL编码119个氨基酸,属于轻链κV家族。实验结果为进一步研究AEG-1与恶性肿瘤发生、发展的关系及在其临床诊断中的应用奠定了基础。

关键词： AEG-1; McAb; 可变区基因; 克隆

Abstract:

Human Astrocyte elevated gene-1 (AEG-1) encodes 582 amino acid residues and locates at chromosome 8q22. AEG-1 involves in multiple signal pathways and represents an important genetic determinant regulating multiple events in tumorigenesis and development. In order to further explore its role in the development of malignant tumors, recombinant AEG-1 protein was used to immunize the BALB/c mice. At the end of immunization, the mouse spleen cells were fused with SP/20 cells to develop hybridoma cells, and after ELISA screening with AEG-1 protein coated plate, an anti-human AEG-1 monoclonal antibody secreting hybridoma cell line was obtained and named as 1E3. The results of Western blot and Immunocellchemistry/Immunohistochemistry detecting showed that this AEG-1 monoclonal antibody could special reacted with AEG-1 protein in cancer cells. Then, the variable region fragment genes of anti-AEG-1 antibody from 1E3 cells were amplified by RT-PCR method, and both the mRNA and protein sequences were analyzed and compared in BLAST, which indentified them as mouse-derived IgG variable heavy and light chains(VH and VL). Moreover, structural analysis of these VH and VL genes were also did by using Kabat online analysis system, and confirmed that both VH (encoding 117 amino acids) and VL (encoding 119 amino acids) genes had complete FWRs and CDRs regions. All these results have perhaps laid a foundation for further study of AEG-1 function in the development of malignant tumors and the potential use of this bio-marker in the clinical diagnosis of malignant tumors.

Key words: AEG-1 McAb Variable Region Clone

收稿日期: 2014-09-28 出版日期: 2014-12-25

ZTFLH:

Q819

基金资助:

国家自然科学基金(81201525)资助项目

通讯作者: 张惠中 E-mail: td_zhz@163.com

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引用本文:

龙敏, 董柯, 王希, 陈曦, 刘冲, 刘丽, 张惠中. 抗人AEG-1单克隆抗体可变区基因克隆及序列分析[J]. 中国生物工程杂志, 2014, 34(12): 10-15.

LONG Min, DONG Ke, WANG Xi, CHEN Xi, LIU Chong, LIU Li, ZHANG Hui-zhong. Developing an Anti-human AEG-1 Monoclonal Antibody Secreting Hybridoma and Its Variable Region Fragment Amplification. China Biotechnology, 2014, 34(12): 10-15.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141202 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I12/10

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