[Cloning and expressing of golgi protein73 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 : 2013-08-01

Jing-Xia Guo, Hong-Shan Wei, Yong-Ji Song, Jing Zhao, Ai-Xia Liu, Jia Liu, Lin Chen, Jun Xu, Bo-An Li, Yuan-Li Mao

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引用次数: 0

Abstract

Objective: To clone and express human Golgi glycoprotein73 protein, and prepare the monoclonal antibody (mAb) against the protein.

Methods: GP73 gene was amplified from HepG2 cells by RT-PCR, then ligated with pQE31 to form recombinant plasmid pQE-GP73 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 was used to detect specificity of mAbs.

Results: The prokaryotic plasmid expressing the recombinant protein was constructed, and the GP73 recombinant protein was expressed and purified. Five hybridoma cell lines that secreted anti-GP73 mAbs were obtained. 2 of 5 mAbs were the IgG1 subtype. Western Blot indicated the mAbs showed specific combination with GP73 protein.

Conclusion: The GP73 recombinant protein is highly purified and has strong antigenicity. The anti-GP73 mAbs were prepared successfully.

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高尔基蛋白73基因片段的克隆表达及重组蛋白单克隆抗体的制备

目的:克隆并表达人高尔基糖蛋白73蛋白，制备抗该蛋白的单克隆抗体(mAb)。方法:采用RT-PCR方法从HepG2细胞中扩增GP73基因，与pQE31连接形成重组质粒pQE-GP73，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GP73重组蛋白。获得5株分泌抗gp73单克隆抗体的杂交瘤细胞株。5个单抗中有2个为IgG1亚型。Western Blot结果表明，单抗与GP73蛋白特异性结合。结论:GP73重组蛋白纯度高，具有较强的抗原性。成功制备抗gp73单克隆抗体。

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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