CAJ | 학술논문

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450 bp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20 000(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1:10 000, 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250 000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FN1 C-末端蛋白,并免疫获得了抗FN1特异性抗体.
목적 구건인섬유련접단백1(FN1)기인적원핵표체재체,유도기표체병순화해단백,제비특이성항체.방법 이용RT-PCR방법 확증인FN1편마서렬450 bp적편단,포함FN1최기단적150개안기산.구건원핵표체질립pRSETA2-FN1,전화대장간균BL21(DE3),FN1단백경이병기-β-D-류대반유당감(IPTG)유도표체.융합단백통과Ni~(2+)-NTA수지친화순화후면역토제비항체혈청.단백질면역인적(Western blot)화면역조화검측기특이성.결과성공구건중조질립pRSETA2-FN1,한제성내절매매절감정급DNA측서균현시삽입편단정학.SDS-PAGE응효현시표체적융합단백상대분자질량약위20 000(FN1-His표첨),여예기결과일치.매련면역흡부시험(ELISA)검측항체효개약위1:10 000, Western blot현시가이특이성지검측출인혈장화간암세포계HepG2전세포렬해액중상대분자질량약250 000적섬유련접단백.면역조직화학가현시FN1재정상폐급폐암조직중적특이성분포.결론 성공지원핵표체료FN1 C-말단단백,병면역획득료항FN1특이성항체.
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 ISO 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 Ni2 + -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 purified 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.