中文摘要：

目的：构建及鉴定胰岛细胞瘤相关蛋白2胞内区（mIA-2i）和IgG Fc嵌合蛋白的真核表达载体pEGFP-N2／mIA-2i-mIgGFc。 方法：实验于2004-09／2005-07在南京医科大学病原生物学系完成。①采用反转录一聚合酶链反应方法从ICR小鼠的脑组织和脾脏中分别扩增胰岛细胞瘤相关蛋白2胞内区（mIA-2i）和IgG Fc段（mIgG Fc），并引入相应的酶切位点（XhoⅠ／BamH Ⅰ和BmH Ⅰ／EcoRⅠ）。②分别将扩增后的片段克隆入pUCm-T质粒，通过XhoⅠ／BamHⅠ双酶切和连接后，获得pUCm-T／mIA-2i-mIgG Fc重组质粒。③经过XhoⅠ／EcoRⅠ双酶切后，将融合基因mIA-2i-mIgG Fc克隆入真核表达载体pEGFP-N2。通过酶切、聚合酶链反应及插入片段序列测定对重组质粒pEGFP-N2／mIA-2i-mIgG Fc进行鉴定。 结果：①从ICR鼠的脑组织和脾脏中反转录-聚合酶链反应获得胰岛细胞瘤相关蛋白2胞内区和IgG Fc段。②通过基因工程操作，获得重组克隆质粒pUCm-T／mIA-2i和pUCm-T／mIgG Fc，并经酶切鉴定克隆成功。③获得重组克隆质粒pUCm-T／mIA-2i-mIgG Fc，经聚合酶链反应和双位点酶切鉴定重组成功。④获得重组质粒pEGFP-N2/mIA-2i-mIgG Fc和pEGFP-N2/mIA-2i，经聚合酶链反应、酶切及测序鉴定证实特异性片断插入真核表达载体成功。 结论：pEGFP-N2／mIA-2i-mIgG Fc真核表达载体的构建．为利用胰岛细胞瘤相关蛋白2基因疫苗预防非肥胖性糖尿病小鼠发生自身免疫性糖尿病的研究奠定了基础。

英文摘要：

AIM： To construct and identify the recombinant eukaryotic expression vector （pEGFP-N2/mIA-2i-mIgG Fc） by intracellular domain of islet cell tumor associated protein-2 （mIA-2i） and IgG Fc chimera （mIgG Fc）. METHODS： The experiment was carried out in the Department of Pathogenic Biology,Nanjing Medical University from September 2004 to July.2005. ①The mIA-2i and mIgG Fc were expended from the brain tissue and spleen of the ICR mice by RT-PCR method to import into the corresponding enzyme sites （Xho Ⅰ / BamH Ⅰ and BamH Ⅰ/EcoR Ⅰ） respectively. ②Two expended fragments were cloned into the pUCm-T vector and the recombinant pUCm-T/mIA-2i-mIgG Fc vector was obtained after Xho Ⅰ/BamH Ⅰ digestion and ligation. ③The chimera gene of mIA-2i-mIgG Fc was cloned into pEGFP-N2 vector after Xho Ⅰ / EcoR Ⅰ digestion, and the recombinant pEGFP-N2/mIA-2i-mIgG Fc vector was identified by endonuelease, PCR and detection of insertion sequence. RESULTS： ①The mIA-2i and mlgG Fc were obtained from the brain tissue and spleen of ICR mice by RT-PCR. ②Recombinant vectors of pUCm- T/mIA-2i and pUCm-T/mlgG Fc were prepared by gene engineering operation and confirmed successfully by endonucleasc. ③Recombinant vector of pUCm-T/mIA-2i-mIgG Fc was prepared by gene combination and confirmed by PCR and dual-site endonuclease. ④ Recombinant vectors of pEGFP-N2/mIA-2i-mIgG Fc and pEGFP-N2/mIA-2i were obtained by gene clone. The insertion of specific DNA into pEGFP-N2/mIA-2i-mIgG Fc was confirmed successfully by endonuclease, PCR and sequencing. CONCLUSION： The construction of eukaryotic expression vector of pEGFP-N2/mIA-2i-mIgG Fc lays the foundation for further study of preventing autoimmune diabetes mellitus in non-obesity diabetes mice through IA-2 gene vaccine.