大鼠sFcγRIIb基因原核表达及活性鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.022

生物技术通报 ›› 2016, Vol. 32 ›› Issue (9): 165-171.doi: 10.13560/j.cnki.biotech.bull.1985.2016.09.022

大鼠sFcγRIIb基因原核表达及活性鉴定

张艳芬¹, 刘利鹏², 陈瑶², 崔哲², 毕克维², 王南南², 刘中成²

1. 河北大学科学技术处,保定 071002; 2. 河北大学药学院,保定 071002

收稿日期:2016-05-06 出版日期:2016-09-25 发布日期:2016-10-10
作者简介:张艳芬,女,硕士,研究方向：生物药学;E-mail：zhangjing@hbu.edu.cn
基金资助:
国家自然科学基金项目（81202338）,河北省自然科学基金项目（H2013201128,H2016201121）,河北省教育厅项目（Z2015013）

Prokaryotic Expression and Activity Identification of Rat sFcγRIIb Gene

ZHANG Yan-fen, LIU Li-peng, CHEN Yao, CUI Zhe, BI Ke-wei, WANG Nan-nan, LIU Zhong-cheng

1. Science and Technology Office of Hebei University,Baoding 071002; 2. College of Pharmaceutical Sciences of Hebei University,Baoding 071002

Received:2016-05-06 Published:2016-09-25 Online:2016-10-10

摘要/Abstract

摘要： 旨在克隆大鼠FcγRIIb基因,构建sFcγRIIb原核表达体系,制备重组大鼠sFcγRIIb蛋白。采用RT-PCR技术从RBL-2H3细胞中克隆FcγRIIb胞外区基因,构建重组表达质粒转化大肠杆菌诱导表达,镍柱亲和层析纯化重组蛋白,复性后利用Western blotting、ELISA进行鉴定。结果显示,成功克隆大鼠sFcγRIIb基因,构建原核表达载体sFcγRIIb-pET17b,转化大肠杆菌BL21（DE3）。通过对表达体系进行优化,确定IPTG浓度为1.0 mmol/L,诱导时间为4 h时蛋白表达效率最高,重组蛋白主要以包涵体形式存在。包涵体经8 mol/L尿素溶解,利用Ni-NTA柱亲和层析获得了较高纯度的重组蛋白。采用梯度透析复性法对sFcγRIIb进行复性后,经Western blotting、ELISA与竞争性ELISA鉴定,重组蛋白sFcγRIIb可被特异性抗体所识别且与IgG具有结合能力。成功建立了大鼠FcγRIIb基因原核表达体系,制备了具有生物学功能的重组蛋白。

关键词: 抑制性受体, Fcγ, RIIb基因, 原核表达, 重组蛋白

Abstract: FcγRIIb is an important inhibitory receptor with immune negative regulation function. The purpose of this study is to clone the rat FcγRIIb gene,construct the prokaryotic expression system of sFcγRIIb,and prepare the recombinant rat sFcγRIIb protein. The extracellular domain gene of FcγRIIb was cloned from RBL-2H3 cells using RT-PCR and the recombinant expression plasmid containing gene sFcγRIIb was constructed and transferred to Escherichia coli. The recombinant protein was purified by nickel column affinity chromatography and identified by Western blotting and ELISA after refolding. As results,the gene sFcγRIIb was successfully cloned,and the prokaryotic expression vector sFcγRIIb-pET17b was constructed and transferred into E. coli BL21（DE3）. By optimizing the expression system,the protein expression efficiency was the highest when the IPTG concentration was 1.0 mmol/L,and the induction time was 4 h. The recombinant proteins were mainly the inclusion bodies and dissolved by 8 mol/L urea. Then the purified recombinant protein was obtained by Ni-NTA column affinity chromatography. The results of Western blotting,ELISA and competitive ELISA showed that the recombinant protein sFcγRIIb after refolding was recognized by specific antibodies and combined with IgG. Conclusively,the prokaryotic expression system of gene sFcγRIIb was successfully established,and the recombinant protein with biological function was prepared.

Key words: inhibitory receptor, gene FcγRIIb, prokaryotic expression, recombinant protein

张艳芬, 刘利鹏, 陈瑶, 崔哲, 毕克维, 王南南, 刘中成. 大鼠sFcγRIIb基因原核表达及活性鉴定[J]. 生物技术通报, 2016, 32(9): 165-171.

ZHANG Yan-fen¹, LIU Li-peng², CHEN Yao², CUI Zhe², BI Ke-wei², WANG Nan-nan², LIU Zhong-cheng². Prokaryotic Expression and Activity Identification of Rat sFcγRIIb Gene[J]. Biotechnology Bulletin, 2016, 32(9): 165-171.

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大鼠sFcγRIIb基因原核表达及活性鉴定

Prokaryotic Expression and Activity Identification of Rat sFcγRIIb Gene

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