[Cloning and expressing of tissue inhibitor of metalloproteinases I gene fragment and preparation of monoclonal antibodies against the recombinant protein].

中华实验和临床病毒学杂志 Pub Date : 2013-06-01

Jun Hou, Jian Zhang, Jing-Xia Guo, Lin Cheng, Jing Zhao, Jia Liu, Jun Xu, Ai-Xia Liu, Yong-Ji Song, Pan-Yong Mao, Bo-An Li, Yuan-Li Mao

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引用次数: 0

Abstract

Objective: To prepare the monoclonal antibody (mAb) against tissue inhibitor of metalloproteinases I (TIMP-I) fusion protein.

Methods: TIMP-I gene was amplified from fibrotic human liver tissue by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-TIMP-I and transformed into E. coli BL21. The protein induced by IPTG was purified by 6 x His-tag and used to immunize the BALB/c mice. The specific monoclonal antibodies (mAbs) were prepared by the cell fusion technique. Western Blot were used to detect specificity of mAbs.

Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the TIMP-I recombinant protein was expressed and purified. Four hybridoma cell lines that secreted anti-TIMP-I mAbs were obtained. 3 of 4 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with TIMP-I protein.

Conclusion: The TIMP-I recombinant protein is highly purified and has strong antigenicity. The anti- TIMP-I mAbs were prepared successfully.

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金属蛋白酶组织抑制剂I基因片段的克隆表达及重组蛋白单克隆抗体的制备

目的:制备抗金属蛋白酶组织抑制剂I (TIMP-I)融合蛋白的单克隆抗体(mAb)。方法:采用RT-PCR方法从人肝纤维化组织中扩增TIMP-I基因，与pQE31连接形成重组质粒pQE-TIMP-I，转化大肠杆菌BL21。IPTG诱导的蛋白经6 × His-tag纯化，用于免疫BALB/c小鼠。通过细胞融合技术制备特异性单克隆抗体(mab)。Western Blot检测单克隆抗体的特异性。结果:构建了表达重组蛋白的原核质粒，表达并纯化了timp - 1重组蛋白。获得4株分泌抗timp - i单克隆抗体的杂交瘤细胞系。4个单抗中有3个为IgG1亚型。Western Blot结果显示，单抗与timp - 1蛋白特异性结合。结论:重组蛋白纯度高，具有较强的抗原性。成功制备了抗TIMP-I单克隆抗体。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

中华实验和临床病毒学杂志

CiteScore

0.20

自引率

0.00%

发文量

4549