人类Foxm1b基因的原核表达、抗体制备及其对肝癌细胞中Foxm1b蛋白的识别

 【目的】构建人类Foxm1b基因的原核表达载体，表达并纯化蛋白，制备多克隆抗体，并用此抗体检测肝癌细胞中Foxm1b基因的表达，为进一步研究Foxm1b基因的功能奠定基础。【方法】从GenBank中下载人类Foxm1b基因全长cDNA序列并用Pegene软件分析其可能的抗原表位，设计引物．应用RT—PCR获得含抗原表位的Foxm1b基因片段，重组人原核表达载体pET-28a，在大肠杆菌BL-21（DE31中诱导表达，应用亲和层析法获得纯度较高的原核表达蛋白并免疫大鼠制备多克隆抗体，用ELISA检测抗体滴度并用免疫细胞化学的方法检测此抗体对肝癌细胞中天然Foxm1b蛋白的识别。【结果】成功构建了Foxm1b基因重组表达载体pET-28a—Foxm1b，表达的蛋白经纯化后免疫大鼠得到了高滴度的多克隆抗体．该抗体可以识别肝癌细胞中的天然抗原。【结论】人类Foxm1b基因的原核表达载体的构建、重组蛋白的表达纯化及有诊断价值的抗体的制备为后续的研究提供了基础。

Prokaryotic Expression of the Human Foxm1b Gene, Preparation of Its Polyclonal Antibody and Application of the Antibody to Recognize the Foxm1b antigen in Liver Tumor Cell

Objective] To construct prokaryotic recombinant plasmid and express Foxm1b protein, prepare its polyclonal antibody and apply it to recognize the nature antigen in liver tumor cell line. Methods] The full cDNA sequence of human Foxm1b gene was obtained from GenBank and analyzed by PCgene bioinformatics software to investigate the antigenic determinants. The sequence containing antigenic determinants of human Foxm1b gene was amplified by RT-PCR and recombined into prokaryotic expression vector pET-28a, then transformed into E.coli BL-21(DE3). After induced by IPTG, the recombinant protein was expressed and purified by affinity purification. The polyclonal antibody was obtained from rats immunized by the recombinant protein and was identified by ELISA. Immuocytochemistry was used to affirm that the polyclonal antibody could recognize the natural Foxm1b protein expressed in liver tumor cell.Results]The recombinant prokaryotic expression vector pET-28a-Foxm1b was constructed and the recombinant protein was purified successfully, and the prepared high titer polyclonal antibody could recognize the natural protein in liver tumor cell very well.Conclusions] The recombinant prokaryotic expression vector, the recombinant protein and the polyclonal antibody obtained successfully in this study would be applied for further investigation of the Foxm1b gene and liver cancer.