摘要
为获得抗人NANOGP8基因的多克隆抗体,采用PCR法扩增了NANOGP8基因,经序列测定正确后,连接到pET-28a质粒上,将获得的重组质粒pET-28a-NANOGP8转化到大肠杆菌BL21(DE3)中,在16℃和37℃下分别进行诱导表达,经SDS-PAGE检测发现融合蛋白以包涵体形式存在于37℃诱导表达的沉淀中,大小约为45kDa。对包涵体进行洗涤、溶解处理,经ChelatingSepharoseFastFlow亲和层析柱进行纯化。对纯化得到的蛋白进行SDS-PAGE及Westernblot免疫印记杂交检测,得到了单一条带。用获得的高纯度融合蛋白免疫新西兰兔,得到兔来源的抗人NANOGP8血清。对抗血清进行蛋白免疫印记杂交反应和酶联免疫吸附反应检测,结果显示成功制备了高滴度和特异性的兔抗人NANOGP8多克隆抗体,与市售抗人Nanog多克隆抗体相比,杂交更迅速,效果更明显。为进一步研究NANOGP8基因在肿瘤发生、发展中的作用奠定了基础。
To obtain polyclonal antibody against human NANOGP8, the NANOGP8 gene was amplified by PCR from the template obtained in our previous work and identified by DNA sequence analysis. Then it was digested by EcoR I and Sal I , and ligated with pET-28a vector which was by the same treatment. Sequenced and blasted with the NCBI GenBank, the recombinant plasmid was named as pET-28a-NANOGP8. The recombinant plasmid was transformed into E. coli BL21 （DE3） and induced by lmmol/L IPTG at 16℃ and 37℃. SDS-PAGE showed that the fusion protein was expressed in the form of inclusion body in the sediment when induced at 37℃. After dissolution and affinity purification with Chelating Sepharose Fast Flow, a single protein band about 45 kDa was identified in the SDS-PAGE and Western blot analysis. One new zealand rabbit was immunized with purified NANOGP8 protein to prepare for the polyclonal antibody against NANOGP8. The NANOGP8 antiserum was obtained and characterized by Western blot, ELISA, and compared with commercial goat anti-human Nanog antibody. Results showed that the antibody had high affinity and specificity, and higher titer compared with commercial anti-human Nanog antibody. The studies provide a favorable tool for further study on relation of tumorigenesis, development and NANOGP8 in the future.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第3期30-35,共6页
China Biotechnology
基金
国家自然科学基金资助项目(30428017)
