【作者】 张健；

【导师】 张万会； 刘新平； 药立波；

【作者基本信息】 中国人民解放军第四军医大学 ， 生理学， 2003， 硕士

【摘要】 人ndrg2基因(N-myc downstream-regulated gene 2)是本课题组于2000年从正常成人全脑cDNA文库中发现和获得的新序列。其全长为2,024bp，经BLAST相似性分析证明所得序列为一新基因(GenBank检索号：AF159092)。ndrg2基因含有16个外显子和15个内含子，定位于染色体14q11.2，编码一个含有357个氨基酸残基、分子量约为40kDa的蛋白质。初步研究显示，该基因可表达于多种组织中，并且是正常脑组织和胶质瘤的差异表达基 第四军医大学硕士学位论文因。目前己报道该家族共有四个成员：ndrgl、ndrgZ、ndrg3禾 ndrg4，其中nd侣4又分为三个亚型：叫＊个B、n巾叶庄“『和叩r＊＊H。珐家族各成员厂的氨基酸残基序列同源性很高，但在各组织间的表达水平有明显差异。 己知有多种因素可以影响 ndrg的表达水平，在施加同型半脐氨酸、致DNA损伤性物质、地衣霉素hunicamycin＼应激刺激（缺氧、Niy等＼抑癌基因p53等因素的条件下，ndrgl 的表达上调；而雄激素、N－myc、C－myc等因素可下调 ndrg 的表达。己有文献报道，在细胞的增箔分化、细胞氧化还原电势平衡的维持、阻抑肿瘤细胞扩散等过程中，ndrg和 ndrgZ发挥着重要作用 ［’””］。由于目前对于 ndrg家族的结构和功能性认识尚不全面，因此，克隆和表达n山g家族各成员并制备相应的特异性抗体则成为迫切而重要的基础性工作，有利于从总体上研究该家族各成员的生理功能。 根据上述事实，本实验按如下方案展厂： 1．ndrg家族成员的cDNA克隆。提取人胎脑组织的总RNA，经反转录制备cDNA。依据GenBanLk登录的ndrg家族各成员序列分别合成了针对ndfgl、ndrg3和 ndfg4B的引物，然后进行 PCR反应以扩增 DNA，回收和纯化PCR产物并将其插入到pMD 181载体中进行克隆后测序分析，用 BLAST程序验证测序结果。 2．ndfg家族成员的原核表达。将测序下确的片段插入到pRSETA。pGEX＊T、pPpoEX卫Tb等表达载体中，分别用 IPTG诱导表达，发现pPROEX－HTbndygl所编码的NDRGI蛋白有高效可溶性表达。 3．NDRGI 蛋白的二级结构分析。融合表达的6HIS．NDRGI 蛋白经NiNTA偶联的琼脂糖珠纯化后，进行圆h色性分析。发现在该分于中，几种蛋白质二级结构所占比列如下：。螺旋：23石9入p片层：18石％，转角：25．7％，无规则卷曲：32刀％，这与计算机程序预测的结果相近。 4．抗人 NDRG家族的多克隆抗体制备。用可溶性的 6HIS－NDRG蛋白 4 第四军医大学硕士学位论文免疫家兔获得了高效价的兔抗人 NDRG多克隆抗血清，利用固定于硝酸纤维素膜上的NDRGI抗原进行亲和吸附，以纯化抗血清，提高了NDRGI多克隆抗体的特异性，并用此纯化的抗体进行了免疫组化和 Western捡测。出于＊＊＊G家族各成员间氨基酸组成有很高的同源性，此抗体在识别＊D＊GI。NDRGZ、NDRG3和 NDRG4时存在交叉，表明己获得抗人NDRG家族的多芜隆抗体。 5．抗人NDRG家族寡肽的多克隆抗体制备。山于抗人NDRG家族的多是隆抗体不能很好地区分NDRG1、NDRGZ、NDRG3和NDRG4，按各亚型分于氦基酸残基序列中特异的肽段合成的寡肽作为半抗原，与KLH偶联后兔疫家兔，以制备特异性的抗寡肽抗体。B前免疫家兔的抗体滴度约为1：3200，继续加强免疫，可望获得具有高度特异性的抗体，以特异地识别NDRGI。NDRGZ、NDRG3和 NDRG4各亚型分子。更多还原

【Abstract】 The human ndrg2 (N-myc downstream regulated gene 2) cDNA was first cloned from normal human whole brain cDNA library in our lab in 2000. The full length of ndrg2 cDNA is 2,024 bp long. BLAST analysis verified that the discovered cDNA sequence represented a new gene (GenBank Accession AF 159092) located on human chromosome 14q11.2. ndrg2 gene consists of 16 exonsand 15 introns that encodes a 40 kDa protein with 357 amino acid residues. Preliminary data indicated that ndrg2 was expressed in multiple tissues, with heterogeneous expression in normal brain structures except glioma specimens lacking ndrg2 expression. Until now the reported four members comprise the ndrg family: ndrgl, ndrg2, ndrg3 and ndrg4, with ndrg4 possessing three isoforms: ndrg4-B, ndrg4-Bvar and ndrg4-H. The four ndrg family members share great homology in their composition of amino acid residues with differential expression in diverse tissues.Many factors might affect the expression of ndrgl gene. The expression of ndrgl is upregulated by homocystein, tunicamycin, DNA damaging agents, stressful stimuli (such as hypoxia and nickel toxication) and p53, and downregulated by androgen and the proto-oncogenes N-myc and c-myc. Existing evidences suggest that ndrgl and ndrg2 play active roles in cell proliferation and differentiation events, maintaining the balance of cell redox potential and suppressing metastasis of malignant tumor cells. As the structure and function of ndrg family genes remain largely unkown, it is very important to firstly clone and express cDNA sequences of ndrg family genes (ndrgl, 3 and 4) coupled with generation of specific antibodies against NDRG proteins. These will greatly facilitate subsequent comprehensive characterization of NDRG functions.Our study were therefore undertaken to address the above issues.1. cDNA cloning of ndrg family genes. Total RNA from human fetal brain tissue were extracted, then reverse transcribed into cDNA. Three pairs of primers were synthesized which target partial sequences in ndrgl, ndrg3 and ndrg4-B according to GenBank deposited sequences. PCR products were inserted into pMD18-T vector and sequenced. Sequences thus obtained were compared toGenBank database using BLAST program.2. The prokaryotic expression of ndrg family. The validated gene fragments were respectively inserted into pRSET-A, pGEX-4T-l and pPROEX-HTb expression vectors. Protein expression was induced by IPTG in E.coli. Bacterial strains that expressed highly soluble 6His-NDRGl in LB medium were obtained by transformation of pPROEX-HTb-ndrgl recombinant plasmid when analyzing their solubility.3. The secondary structural analysis of NDRG 1 fusion protein. The expressed fusion protein was purified by Ni-NTA agarose beads affinity chromatography. The secondary structure of purified 6His-NDRGl fusion protein was analyzed by circular dichroism. The results indicated that 6His-NDRGl was composed of a -Helix:23.6%,P-sheet:18.6%, Turn:25.7%, Random:32.0%. The outcome of analysis basically conforms to predicted data using computer programs.4. Polyclonal antibody preparation against NDRG family. Immunization of rabbits with fusion protein produced high titer polyclonal antibodies against NDRG1. The specific antibody was purified by absorbing antiserum with NDRG1 antigen immobilized on NC filters. Satisfactory results were obtained in immunohistochemical staining and Western blot using the generated antibodies. Due to the high homology in composition of amino acids residues in predicted ndrg family proteins, the antibody prepared against NDRG1 antigen failed to differentiate the four members of NDRG family. Thus an antibody against all NDRG family proteins was obtained.5. Polyclonal antibody preparation against the peptides of NDRG family proteins. We synthesized four short peptides as haptens that represent unique segments in protein sequences of NDRG1, NDRG2, NDRG3 and NDRG4respectively. These peptides conjugated with Keyhole limpet hemocyanin (KLH) were recently u更多还原