B细胞特异激活蛋白基因Pax5原核表达、纯化及多克隆抗体制备研究  

Prokaryotic expression,purification,and polyclonal antibody preparation of Pax5,an B-cell specific activator protein encoding gene

作　　者：王旭[1] 逄越[1] 王惠国[1] 马彪[1] 刘庆平[1] 李文哲[1] 

机构地区：[1]大连大学生物工程学院免疫研究所,116622

出　　处：《免疫学杂志》2011年第6期519-522,543,共5页Immunological Journal

基　　金：国家自然科学基金(30972675);大连市科技计划项目(2010J21DW011)

摘　　要：目的克隆人B细胞特异激活蛋白基因Pax5,并实现Pax5的原核表达、纯化及多克隆抗体制备。方法从人淋巴瘤细胞株Raji提取总RNA,用RT-PCR扩增Pax5基因片段,克隆至pMD19-T载体,双酶切及基因测序鉴定后,再定向插入原核表达载体pET28a中,并转化E.coli BL21(DE3),IPTG诱导表达目的蛋白,进行SDS-PAGE分析,纯化后进行Western blot分析,用原核表达蛋白制备兔抗Pax5抗血清。利用免疫荧光染色法观察Raji细胞中的Pax5表达情况。结果克隆得到的目的基因片段大小与预期相符,测序结果与GenBank中报道的序列完全一致,成功构建了重组表达质粒pET28a-Pax5,并获得目的蛋白。Western blot检测原核表达Pax5蛋白相对分子质量约为42 000,经镍离子金属螯合柱纯化后,纯度达95%以上。兔抗Pax5抗血清ELISA效价可达1∶256 000。免疫荧光染色结果显示Raji细胞中Pax5高表达。结论本研究在原核系统中成功表达了Pax5重组蛋白,并制备多克隆抗体,为开发B淋巴细胞白血病诊断试剂盒提供了实验数据。This study aimed to construct a prokaryotic expression plasmid pET28a-Pax5,express the encoded B-cell specific activator protein（BSAP）,and prepare its polyclonal antibody.Total RNA was extracted from Raji cells,and then the cDNA encoding BSAP was subcloned into pMD19-T vector.The intact region encoding Pax5 was cloned into a prokaryotic expression vector pET-28a（＋） to yield pET-28a（＋）-Pax5,which was then highly expressed in Escherichia coli（E.coli） BL21（DE3） cells under 0.5 mmol/L IPTG induction.The BSAP expressed in E.coli strain BL21 was analyzed by SDS-PAGE.After purification by nickel affinity chromatography,the recombinant c-Myc protein was used to raise rabbit anti Pax5 antibody.The enzyme-linked immunosorbent assay（ELISA） showed titer of rabbit anti BSAP antibody was 1∶256 000,with a high sensitivity.While immunocytochemistry demonstrated that the nuclear localization of BSAP was up-regulated in Raji cells.All this data indicated that the anti pax5 antibody is generated successfully,which would provide a reliable foundation for establishing a diagnostic method of B lymphoma.

关 键 词：PAX5 BSAP 原核表达 

分 类 号：R392.11[医药卫生—免疫学]