纤维连接蛋白1的原核表达、纯化及抗体的制备  

Prokaryotic expression,purification and antibody preparation of human fibronectin 1

作　　者：王菲菲[1] 苏畅[1] 吴海东[1] 王萌[1] 孔鹏洲[1] 刘民[1] 李欣[1] 汤华[1] 

机构地区：[1]天津医科大学基础医学院天津市生命科学中心实验室,300070

出　　处：《国际免疫学杂志》2010年第1期60-64,共5页International Journal of Immunology

基　　金：基金项目：国家高技术研究发展计划（863）（2007AA021808）;国家自然科学基金资助项目（30873017）;天津市自然科学基金重点项目（08JCZDJC23300,09JCZDJC17500）

摘　　要：目的构建人纤维连接蛋白1（FN1）基因的原核表达载体，诱导其表达并纯化该蛋白，制备特异性抗体。方法利用RT—PCR方法扩增人FN1编码序列450bp的片段，包含FN1羧基端的150个氨基酸。构建原核表达质粒pRSETA2-FN1，转化大肠杆菌BL21（DE3），FN1蛋白经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。融合蛋白通过Ni^2＋-NTA树脂亲和纯化后免疫兔制备抗体血清。蛋白质免疫印迹（Western blot）和免疫组化检测其特异性。结果成功构建重组质粒pRSETA2-FN1，限制性内切酶酶切鉴定及DNA测序均显示插入片段正确。SDS—PAGE凝胶显示表达的融合蛋白相对分子质量约为20000（FN1-His标签），与预期结果一致。酶联免疫吸附试验（ELISA）检测抗体效价约为1：10000，Western blot显示可以特异性地检测出人血浆和肝癌细胞系HepG2全细胞裂解液中相对分子质量约250000的纤维连接蛋白。免疫组织化学可显示FN1在正常肺及肺癌组织中的特异性分布。结论成功地原核表达了FN1C-末端蛋白，并免疫获得了抗FN1特异性抗体。Objective To express human fibronectin 1 and generate specific human fibronectin 1 antibody in rabbit. Methods A 450 bp length fragment of human FN1 gene, containing the 150 amino acids of the C-terminal, was amplified by RT-PCR, and was cloned to pRSETA2 vector. The expression of FN1 was in BL21 （DE3） and the protein was purified by the Ni^2 ＋-NTA resin. Purified FN1 was injected to the rabbit to raise antibody, and purified the antibody by affinity method. The specification of the antibody was detected through Western blot and immunohistochemistry. Results The pRSETA2 -FN1 vector was confirmed correctly through restriction enzyme digestion and sequencing. The fusion protein with 20 000 （ FN1-His tag） was puri- fied by Ni^2＋ -NTA column. The titer of generated FN1 antibody in rabbit was about 1：10 000 by ELISA. The purified FN1 antibody by affinity was used to detect the FN1 in lung and lung cancer tissue by immunohistochemistry, and to detect FN1 in human plasma and HepG2 cell lysate by western blot. Conclusion The C-terminal of human FN1 was successfully prokaryotically expressed in this study. A specific FN1 antibody was generated by this recombinant FN1 protein.

关 键 词：纤维连接蛋白 基因表达 抗体 癌症 

分 类 号：R3[医药卫生—基础医学]