Prokaryotic Expression, Purification Of Nanog Gene And Preparation Of Antibody

Embryonic stem cells (ES cells) are derived from cells of early embryo and in vitro culturing of these cells can finally maintain the characteristics of self-renewal and pluripotency on development. Understanding the system of transcription control to ES cells is the foundation on understanding the process of human development and measuring cell therapy. The homeobox transcription factors play a important role in differentiation of a good many biologic cells. The Nanog gene is a homeobox transcription factor founded in 2003, the expression of which play an essential role in maintaining self-renewal and pluripotency of ES cells.At present, there are only few reports of Nanog gene expressed in prokaryotic cells and preparation of antibody. In our lab, the whole ORF of Nanog gene was cloned from mouse ES-D3 by RT-PCR. In our research, the mouse Nanog gene was subcloned by PCR, and the prokaryotic expression vector contained the target gene of Nanog gene was constructed, transformed into Escherichia coli BL21(DE3). The Nanog fusion protein was expressed and purified, which provide the excellent tools for further detecting its function. Meanwhile, The purified Nanog protein from E.coli. BL21( DE3) acts as immuneogen to prepare polyclonal antibody. These offer powerful base for the research of its orientation in ES cells and the mechanism of its function. The research works are as follows:1. PCR primers with the sites of double restriction enzyme digestion, BamH I and HindⅢwere designed according to mouse Nanog gene sequence deposited in GenBank and to plasmid pET-32a vector map. Based on the recombinant plasmid pNA992 with target gene of mouse Nanog, the target fragment was obtained by PCR amplification.2. The purified PCR product was digested with both BamH I and HindⅢ, and the Nanog gene fragment was recycled. The Nanog gene digestion fragment was ligated with the pET-32a, which had been digested by the same enzymes. The ligated products were transformed into E.coli. TG I, positive recombinant plasmid was selected on the media contained ampicillin, and then further identified by colony PCR, endoenzyme digestion and sequencing. Positive clone was named as pTE-32a-Nanog. 3. Recombinant plasmid DNA pET-32a-Nanog was extracted and transformed into E.coli. BL21(DE3). After induction by Isopropy1-β-D-thiogalactoside (IPTG), there was a specia fusionl protein band of approximately 57 KDa, and the molecular mass of the tag was 20 KDa. The results showed that the molecular mass of Nanog protein was 37 KDa, which was consistent with those of the previous reports. The optimum condition for the expression of Nanog fusion protein was induced with 0.01mmol/L IPTG for 3 h by comparing the expression result of different IPTG density, cell induced culture times.4. Expression form of fusion protein was analyzed, and the result showed that the recombinant protein was in the form of inclusion body. The inclusion body was washed and dissolved, then the Nanog protein with 6his-tag was purified by HisTrap HP column, and renatured by gradual dialysis, and concentrated. The samples were analyzed by SDS-PAGE on 12% gels. The result showed that the purification of Nanog fusion protein was up to 97%.5. Purified Nanog fusion protein was injected into rabbits to produce a polyclonal antibody to Nanog. Titer of anti-Nanog antibody was upwards 1:64000 by indirect ELISA. The results of Western blot showed that the serum (diluted to 1:10000) had specific antigen-antibody recognition to recombinant Nanog protein.6. The own Anti-Nanog McAb and standard antibody were used for studying immunocytochemical analysis on Nanog protein of ES cells, respectively. The result showed that the own antibody was equal to standard antibody, can specially react to Nanog protein of ES cells, and the best concentration was diluted to 1:2000, the standard was 1:500. So the own antibody can specially react to natural Nanog protein and the titer was higher.