【作者】 李军；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2007， 硕士

【摘要】 胚胎干细胞(Embryonic Stem Cells, ES细胞)起源于早期胚胎细胞,培养扩增时,可维持未分化状态并具有自我更新能力的原始细胞。理解ES细胞多能性和自我更新的转录调控系统是理解人类发育过程及实现这些细胞治疗潜能的基础。其中同源域转录因子在进化上保守且在很多生物的细胞分化中起关键性作用。Nanog是2003年才发现的一个同源域转录因子,它的表达在维持ES细胞上的多能性及自我更新中有重要作用。目前,国内外关于Nanog原核表达及抗体制备的研究报道几乎没有。本实验室研究人员已经通过RT-PCR技术从小鼠ES-D3中克隆获得了完整的Nanog开放阅读框(Open Reading Frame, ORF)。本课题是通过PCR亚克隆小鼠Nanog基因,构建原核表达载体,转化大肠杆菌BL21(DE3)表达Nanog融合蛋白并纯化,为该基因体外功能的进一步验证提供依据。同时将获得的融合蛋白作抗原制备多克隆抗体,为目的基因产物在ES细胞内的定位以及其作用机理研究奠定基础。研究工作取得以下进展:1.根据小鼠Nanog基因的序列和原核表达载体pET-32a的酶切位点,设计两条分别带有BanH I和HindⅢ酶切位点的引物,以带有小鼠Nanog基因的质粒pNA992为模板,利用PCR方法扩增获得一个长度与Nanog基因完整片段相当的特异性片段。2.采用限制性内切酶BamH I和HindⅢ双酶切含酶切位点的目的片段,回收并纯化目的片段Nanog,把该片段连接到经BamH I和HindⅢ限制性内切酶酶切的表达载体pET-32a上,转化E.coli菌株TGⅠ,氨苄青霉素筛选抗性菌落,经菌落PCR、双酶切及测序进一步鉴定获得转化子pET-32a-Nanog。3.将重组表达载体pET-32a-Nanog转化到高效表达菌株BL21(DE3),诱导培养后,重组菌特异性表达了大小为57 kDa的融合蛋白,而空载体表达的特异性的蛋白大小为20 KDa,表明Nanog蛋白的大小为37 KDa,与前人报道一致。通过不同诱导时间及不同诱导剂浓度的对照实验,确定了最佳诱导条件是37℃,0.01 mM IPTG诱导表达3h。4.对融合蛋白的表达形式分析后显示,Nanog融合蛋白以包涵体形式表达。对包涵体洗涤、变性溶解后,采用His Trap HP亲和层析柱纯化Nanog基因所表达的带有6个His-Tag的蛋白并透析复性后浓缩,SDS-PAGE电泳检测纯化的蛋白样品。结果显示其蛋白的纯度达到97%。5.用纯化的Nanog融合蛋白免疫健康的雄性家兔,制备多克隆抗体。间接ELASA法测定多克隆抗体的效价为1: 64000以上。血清1:10000稀释后,Western blot检测表明获得的抗体特异性好,可以特异性的识别重组Nanog蛋白。6.用自制抗体与标准抗体分别对ES细胞进行免疫细胞化学染色,结果显示,自制抗体与标准抗体一样能特异性识别ES细胞中的Nanog蛋白,且其最佳稀释浓度为1:2000,而标准抗体为1:500。表明自制抗体可以特异性识别天然的Nanog蛋白且效价较高。更多还原

【Abstract】 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.更多还原