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1：3
200，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。
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 exons
and 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 to
GenBank 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 NDRG4
respectively. These peptides conjugated with Keyhole limpet hemocyanin (KLH) were recently u
引文
1. Stanton BR, Perkins AS, Tessarollo L, Sassoon DA, Parada LF. Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. Genes Dev, 1992; 6(12A): 2235-47
2. Charron J, Malynn BA, Fisher P, Stewart V, Jeannotte L, Goff SP, Robertson EJ, Alt FW. Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene. Genes Dev, 1992; 6(12A): 2248-57
3. Shimono A, Okuda T, Kondoh H. N-myc-dependent repression of ndrl, a gene identified by direct subtraction of whole mouse embryo cDNAs between wild type and N-myc mutant. Mech Dev, 1999; 83(1-2): 39-52
4. Okuda T, Kondoh H. Identification of new genes ndr2 and ndr3 which are related to Ndr1/RTP/Drg1 but show distinct tissue specificity and response to N-myc. Biochem Biophys Res Commun, 1999; 266(1): 208-15
5. Yamauchi Y, Hongo S, Ohashi T, Shioda S, Zhou C, Nakai Y, Nishinaka N, Takahashi R, Takeda F, Takeda M. Molecular cloning and characterization of a novel developmentally regulated gene, Bdm1, showing predominant expression in postnatal rat brain. Brain Res Mol Brain Res, 1999; 68(1-2): 149-58
6. Nishinaka N, Hongo S, Zhou CJ, Shioda S, Takahashi R, Yamauchi Y, Ohashi T, Ohki T, Nakada N, Takeda F, Takeda M. Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain. Brain Res Der Brain Res, 2000; 120(1): 57-64
7. Kokame K, Kato H, Miyata T. Homocysteine-respondent genes in vascular
endothelial cells identified by differential display analysis GRP78/BiP and novel genes. J Biol Chem, 1996; 271(47): 29659-65
8. van Belzen N, Dinjens WN, Diesveld MP, Groen NA, van der Made AC, Nozawa Y, Vlietstra R, Trapman J, Bosman FT. A novel gene which is up-regulated during colon epithelial cell differentiation and down-regulated in colorectal neoplasms. Lab Invest, 1997; 77(1): 85-92
9. Kurdistani SK, Arizti P, Reimer CL, Sugrue MM, Aaronson SA, Lee SW. Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage. Cancer Res, 1998; 58(19): 4439-44
10. Zhou D, Salnikow K, Costa M. Cap43, a novel gene specifically induced by Ni~(2+) compounds. Cancer Res, 1998; 58(10): 2182-89
11． 邓艳春，药立波，刘新平，聂小燕，王吉村，张小光，苏成芝．一种含有ACP样结构域新基因的发现．生物化学与生物物理进展，2001；28(1)：72-76
12. Zhou RH, Kokame K, Tsukamoto Y, Miyata T. Characterization of the human NDRG gene family: a newly identified member, NDRG4, is specifically expressed in brain and heart. Genomics, 2001; 73(1): 86-97
13. Zhao W, Tang R, Huang Y, Mao Y. Cloning and expression pattern of the human NDRG3 gene. Biochim Biophys Acta, 2001; 1519(1-2): 134-38
14. Qu X, Zhai Y, Wei H, Zhang C, Xing G, Yu Y, He F. Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family. Mol Cell Biochem, 2002; 229(1-2): 35-44
15. Lachat P, Shaw P, Gebhard S, Van Belzen N, Chaubert P, Bosman FT. Expression of NDRG1, a differentiation-related gene, in human tissues. Histochem Cell Biol, 2002; 118(5): 399-408
16. Guan RJ, Ford HL, Fu Y, Li Y, Shaw LM, Pardee AB. Drg-1 as a
differentiation-related, putative metastatic suppressor gene in human colon cancer. CancerRes, 2000; 60(3): 749-55
17. Kalaydjieva L, Gresham D, King RH, Thomas PK. N-myc downstream-regulated gene 1 is mutated in hereditary motor and sensory neuropathy-Lom. Am J Hum Genet, 2000; 67(1): 47-58
18. Ohki T, Hongo S, Nakada N, Maeda A, Takeda M. Inhibition of neurite outgrowth by reduced level of NDRG4 protein in antisense transfected PC12 cells. Brain Res Dev Brain Res, 2002; 135(1-2): 55-63
19. Salnikow K, Blagosklonny MV, Ryan H, Johnson R, Costa M. Carcinogenic nickel induces genes involved with hypoxic stress. Cancer Res, 2000; 60(1): 38-41
20. Salnikow K, Kluz T, Costa M. Role of Ca~(2+) in the regulation of nickel-inducible Cap43 gene expression. Toxicol Appl Pharmacol, 1999; 160(2): 127-32
21. Shaw E, McCue LA, Lawrence CE, Dordick JS. Identification of a novel class in the alpha/beta hydrolase fold superfamily: the N-myc differentiation-related proteins. Proteins, 2002; 47(2): 163-68
22. Salnikow K, Kluz T, Costa M, Piquemal D, Demidenko ZN, Xie K, Blagosklonny MV. The regulation of hypoxic genes by calcium involves c-Jun/AP-1, which cooperates with hypoxia-inducible factor 1 in response to hypoxia. Mol Cell Biol, 2002; 22(6): 1734-41
23．张文红，刘新平，邓艳春，药立波．兔抗人NDR2高效价抗血清的制备、纯化及鉴定．第四军医大学学报，2002；23(8)：716-19
24．李剑，刘新平，邓艳春，孟庆军，药立波．生物化学与生物物理进展，2002；29(2)：223-27
25．曾益新．肿瘤学 北京：人民卫生出版社，1999
26. Harlow E, Lane D. Antibody (A laboratory manual). Cold Spring Harbor laboratory,1988
27. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A Laboratory Manual,2nd ed. Cold Spring Harbor Laboratory Press, 1989; 366～67
28. Chomzynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extracition. Anal Biochem, 1987;162:156-59
29. Marshak DR, Kadonaga JT, Burgess RR, Knuth MW, Brennan WA, Lin SH. Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Beijing, Science Press, 1999
30. Xie Y, Wetlaufer DB. Control of aggregation in protein refolding: the temperature-leap tactic. Protein Sci, 1996; 5(3): 517-23
31. Georgiou G, Valax P. Expression of correctly folded proteins in Escherichia coli. Curr Opin Biotechnol, 1996; 7(2): 190-97
32. Marston F A. The purification of eukaryotic polypeptides synthesized in E.coli. Biochem J, 1986; 240(1): 1-12
33. Bradford MM. A Rapid and sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Priciple of Protein-Dye Binding. Anal Biochem, 1976; 72: 248-54
34. F．奥斯伯等著；颜子颖，王海林译，精编分子生物学实验指南。北京，科学出版社，1999
35. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning (A Laboratory Course Manual, 2nded). Beijing, Science Press, 1996
36. Li YM, Zhao YQ. Practical Protocols in Molecular Biology, Beijing, Science Press, 1998
37. Nunez E, Wei X, Delgado C, Rodriguez-Crespo I, Yelamos B. Cloning, expression,and purification of histidine-tagged pres domains of hepatitis B virus. Protein
Expr Purif, 2001;21(1): 183-91
38. Carlsson J, Drevin H, Axen R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem J, 1978; 173(3): 723-37
39. Neurath AR, Strick N. Enzyme-linked fluorescence immunoassays using beta-galactosidase and antibodies covalently bound to polystyrene plates. J Virol Methods, 1981; 3(3): 155-65
40． 何畏、梁志清、吴玉章、李玉清．一种精子蛋白嵌合肽的实验研究．中华医学杂志，2002；82(7)：484—86．
41． 高丽华、胡显文、张正光．免疫亲和层析法纯化单链尿激酶型纤溶酶原激活剂．生物工程学报，2002；18(3)：356—59