基础研究 Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-02-15; 12(2): 336-338
在线出版日期: 2004-02-15. doi: 10.11569/wcjd.v12.i2.336
人肝素酶基因正反义腺病毒表达载体的构建及鉴定
蔡永国, 房殿春, 杨仕明, 罗元辉, 杨孟华, 王东旭
蔡永国, 房殿春, 杨仕明, 罗元辉, 杨孟华, 王东旭, 中国人民解放军第三军医大学西南医院全军消化中心 重庆市 400038
蔡永国, 男, 1970-11-11生, 山东沂水人, 汉族. 博士研究生, 发表论文6篇, 主要从事消化道肿瘤的研究
基金项目: 国家自然科学基金资助项目, No. 30200123.
通讯作者: 杨仕明, 400038, 重庆市, 中国人民解放军第三军医大学附属西南医院全军消化专科中心. shimingyang@yahoo.com
电话: 023-68754000-73057
收稿日期: 2003-08-08
修回日期: 2003-09-10
接受日期: 2003-09-24
在线出版日期: 2004-02-15

目的: 构建人肝素酶正义和反义腺病毒表达载体.

方法: 用EcoR Ⅰ从pcDNA3-hpa质粒上切下约1.7 kb的人肝素酶全长cDNA片段, 然后连入pDC315质粒的EcoRⅠ酶切位点上, 经BamH Ⅰ酶切鉴定出正义和反义表达载体, 并对正义重组质粒进一步采用测序鉴定其方向性.

结果: 经BamH Ⅰ酶切后, 正义质粒形成4.3 + 1.4 kb两条带, 而反义重组质粒为5.1 + 0.4 kb两条带, 与理论计算值完全一致; 测序结果与Gene Bank报告的肝素酶序列完全一致.

结论: 成功构建了人肝素酶的正、反义腺病毒表达载体, 为进一步研究正、反义肝素酶基因转染对肿瘤细胞的影响奠定了基础.

关键词: N/A
引文著录: 蔡永国, 房殿春, 杨仕明, 罗元辉, 杨孟华, 王东旭. 人肝素酶基因正反义腺病毒表达载体的构建及鉴定. 世界华人消化杂志 2004; 12(2): 336-338
Construction and identification of sense and antisense human heparanase adenovirus expression vector
Yong-Guo Cai, Dian-Chun Fang, Shi-Ming Yang, Yuan-Hui Luo, Meng-Hua Yang, Dong-Xu Wang
Yong-Guo Cai, Dian-Chun Fang, Shi-Ming Yang, Yuan-Hui Luo, Meng-Hua Yang, Dong-Xu Wang, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
Supported by: the National Natural Science Foundation of China, No. 30200123.
Correspondence to: Dr. Shi-Ming Yang, Department of Gastroenterology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. shimingyang@yahoo.com
Received: August 8, 2003
Revised: September 10, 2003
Accepted: September 24, 2003
Published online: February 15, 2004

AIM: To construct an adenovirus expressing vector of sense and antisense human heparanase gene.

METHODS: The human heparanase cDNA fragment contained in the pcDNA3-hpa vector was cloned into the adenovirus expressing vector pDC315 in cis-direction or trans-direction using DNA recombinant technology. The recombinant vectors were identified by digestion of BamH I. The sense recombinant vector was further identified by DNA sequencing.

RESULTS: After digested by BamH I, two fragments which lengthened 4.3 and 1.4 kb were formed in sense recombinant vector (pDC315-sHpa), while two fragments which lengthened 5.1 kb and 0.4 kb were formed in antisense vector (pDC315-aHpa). Electrophoresis results were completely coincident with theoretical calculation. pDC315-sHpa DNA sequence was identical to the heparanase sequence published in the Gene Bank.

CONCLUSION: The sense and antisense human heparanase adenovirus expressing vectors are successfully constructed.

Key Words: N/A
0 引言

肝素酶是近年来克隆成功的与肿瘤转移相关的一种葡萄糖苷内切酶, 其编码基因定位于4q21.3, 1999年最先由Voldavsky et al(Nat Med 1999; 5: 793)和Hulett et al(Nat Med 1999; 5: 803)克隆鉴定. 肝素酶能够降解细胞外基质和基底膜的主要组成成分硫酸肝素蛋白聚糖(HSPG), 从而促进肿瘤细胞的浸润与转移. 肝素酶成为肿瘤转移治疗的一个新的基因靶点. 我们通过构建人肝素酶正反义腺病毒表达载体, 为进一步研究肝素酶对肿瘤转移细胞的生物学影响创造了条件.

1 材料和方法
1.1 材料

DH5α大肠杆菌菌株由本室保存; pDC315腺病毒质粒由第三军医大学全军免疫研究中心邹强博士惠赠; 含人全长肝素酶cDNA的pcDNA3-Hpa质粒由澳大利亚学者Parish 及Hulett教授惠赠; 限制性内切酶EcoRⅠ, BamHⅠ和牛小肠碱性磷酸酶(CIP)购自美国New England Biolabs公司; 质粒提取试剂盒购自美国Omega公司; 琼脂糖DNA回收试剂盒购自Roche公司.

1.2 方法

肝素酶正、反义腺病毒表达载体的构建及初步鉴定策略见图1.

图1
图1 肝素酶正、反义腺病毒表达载体的构建及鉴定策略.

pcDNA3-Hpa与pDC315质粒的转化采用氯化钙法, 质粒提取采用Omega公司的质粒提取试剂盒, 并用EcoRⅠ酶切鉴定; 肝素酶全长1.72 kb, 克隆在质粒pcDNA3-hpa的EcoRⅠ酶切位点, 用EcoRⅠ酶切该质粒, 8 g/L琼脂糖电泳, 然后用琼脂糖DNA回收试剂盒回收含肝素酶 cDNA的DNA片段(1.72 kb), 溶于去离子水中, -20 ℃冻存备用; 用EcoRⅠ酶切pDC315质粒使其线性化, 试剂盒回收片段, 牛小肠碱性磷酸酶去磷酸化, 然后酚氯仿抽提, 无水乙醇沉淀, TE缓冲液溶解, -20 ℃保存备用; 2.5 μL去磷酸化的pDC315/EcoRⅠ与2.5 μL 肝素酶 cDNA片段混合均匀, 45 ℃反应5 min, 迅速转移到冰上, 加T4 DNA连接酶1 μL混匀, 4 ℃过夜进行连接反应; 将连接产物用氯化钙法转化大肠杆菌, 将200 μL转化产物涂于含氨苄青霉素的LB培养板上, 37 ℃过夜. 次日挑取数个菌落置于含氨苄青霉素的LB液体培养基中, 37 ℃振摇过夜, 质粒提取试剂盒提取质粒, 与空载体一起用8 g/L琼脂糖凝胶电泳筛选出重组质粒, 然后按重组质粒图谱, 用BamHⅠ限制性内切酶进行酶切鉴定. 将BamH I鉴定正确的正义肝素酶重组子交上海生物工程公司进行基因测序以进一步鉴定连接的正确性.

2 结果

如图2, 图3所示: pcDNA3-Hpa质粒经EcoRⅠ酶切后, 形成5.4 kb和1.7 kb两个电泳条带, 试剂盒回收1.7 kb的电泳条带; pDC315腺病毒载体经EcoRⅠ酶切后, 形成3.9 kb一条带, 片段经试剂盒回收、CIP去磷酸化后, 与肝素酶片段相连接, 连接产物转化大肠杆菌后, 得到大量阳性菌落, 随机挑取数个菌落小提质粒, 琼脂糖凝胶电泳筛选出重组体, 然后用BamHⅠ酶切鉴定, 正义重组质粒为4.3+1.4 kb两条带, 而反义重组质粒为5.3+0.4 kb两条带, 与理论计算完全符合. 基因测序结果表明全长人肝素酶cDNA序列正确接入到重组质粒中, 如图4所示. 证明含有人肝素酶的正、反义腺病毒表达载体构建成功, 分别命名为pDC315-sHpa和pDC315-aHpa.

图2
图2 pcDNA3-hpa和pDC315经EcoR I酶切鉴定. 1: Marker(λDNA/Hind III + EcoR I); 2: pcDNA3-Hpa; 3: pcDNA3-HPA/EcoRⅠ; 4: pDC315; 5: pDC315/EcoRⅠ.
图3
图3 肝素酶正、反义腺病毒表达载体的鉴定. 1: Marker(λDNA/Hind III + EcoR I); 2、3: 肝素酶重组子; 4: 肝素酶正义载体; 5: 肝素酶反义载体.
图4
图4 正义肝素酶表达质粒pDC315-sHpa DNA序列(局部).
3 讨论

硫酸乙酰肝素蛋白多糖(heparin sulphate proteoglycan HSPG)是组成细胞外基质和基底膜的重要组成成分. 肝素酶是一种葡糖苷酸内切酶, 他能够裂解连接于硫酸乙酰肝素蛋白多糖核心分子上的硫酸肝素, 破坏细胞外基质和基底膜结构的完整性, 在促进肿瘤细胞的浸润及转移过程中发挥重要作用.

肝素酶基因于1999年被克隆成功后, 现已明确肝素酶基因位于人染色体4q21.3[1-2]. 之后研究发现在人类多种肿瘤组织中均有肝素酶的表达, 而且肝素酶的表达水平与恶性肿瘤细胞的转移潜能相关, 并与肿瘤患者的预后相关[3-19]. 在高转移的鼠腺癌细胞系中肝素酶mRNA高表达, 而在低转移或不转移的细胞系中只有极少量表达[20]. 高转移潜能的肿瘤细胞的肝素酶活性比低转移潜能的肿瘤细胞高4-10倍, 无转移潜力的鼠淋巴瘤和低转移潜力的黑色素瘤细胞系导入肝素酶基因后, 均获得了高转移潜力[21]. 临床研究发现反义介导抑制人肝素酶基因表达可以抑制人癌细胞的胸膜播散[22], 通过抑制肝素酶活性可以抑制肿瘤细胞的转移[23-26], 目前肝素酶的抑制剂PI-88已经进入II期临床试验.

肝素酶促进肿瘤细胞转移的机制除通过降解HSPG, 破坏细胞外基质和基底膜, 促进肿瘤细胞的侵袭与转移外, 还通过促进HS结合性生长因子或细胞因子释放, 间接促进肿瘤细胞的转移[27].

研究表明胃癌组织肝素酶阳性率为80%左右[5,28-29], 由于在大多数胃癌组织中肝素酶均有表达, 肝素酶可以成为针对胃癌转移的基因治疗的良好靶位. 采用分子克隆技术, 构建腺病毒表达载体是目前基因治疗的常用方法[30-32], 我们将人全长肝素酶cDNA导入到腺病毒载体pDC315中, 成功构建了人肝素酶正、反义腺病毒表达载体, 为进一步研究肝素酶基因转染对肿瘤细胞生物学行为的影响奠定了基础.

编辑: N/A

1.  Dong J, Kukula AK, Toyoshima M, Nakajima M. Genomic organization and chromosome localization of the newly identified human heparanase gene. Gene. 2000;253:171-178.  [PubMed]  [DOI]
2.  Baker E, Crawford J, Sutherland GR, Freeman C, Parish CR, Hulett MD. Human HPA endoglycosidase heparanase. Map position 4q21.3. Chromosome Res. 1999;7:319.  [PubMed]  [DOI]
3.  Marchetti D, Li J, Shen R. Astrocytes contribute to the brain-metastatic specificity of melanoma cells by producing heparanase. Cancer Res. 2000;60:4767-4770.  [PubMed]  [DOI]
4.  Rohloff J, Zinke J, Schoppmeyer K, Tannapfel A, Witzigmann H, Mössner J, Wittekind C, Caca K. Heparanase expression is a prognostic indicator for postoperative survival in pancreatic adenocarcinoma. Br J Cancer. 2002;86:1270-1275.  [PubMed]  [DOI]
5.  Endo K, Maejara U, Baba H, Tokunaga E, Koga T, Ikeda Y, Toh Y, Kohnoe S, Okamura T, Nakajima M. Heparanase gene expression and metastatic potential in human gastric cancer. Anticancer Res. 2001;21:3365-3369.  [PubMed]  [DOI]
6.  Maxhimer JB, Quiros RM, Stewart R, Dowlatshahi K, Gattuso P, Fan M, Prinz RA, Xu X. Heparanase-1 expression is associated with the metastatic potential of breast cancer. Surgery. 2002;132:326-333.  [PubMed]  [DOI]
7.  Zcharia E, Metzger S, Chajek-Shaul T, Friedmann Y, Pappo O, Aviv A, Elkin M, Pecker I, Peretz T, Vlodavsky I. Molecular properties and involvement of heparanase in cancer progression and mammary gland morphogenesis. J Mammary Gland Biol Neoplasia. 2001;6:311-322.  [PubMed]  [DOI]
8.  Ikuta M, Podyma KA, Maruyama K, Enomoto S, Yanagishita M. Expression of heparanase in oral cancer cell lines and oral cancer tissues. Oral Oncol. 2001;37:177-184.  [PubMed]  [DOI]
9.  Mikami S, Ohashi K, Usui Y, Nemoto T, Katsube K, Yanagishita M, Nakajima M, Nakamura K, Koike M. Loss of syndecan-1 and increased expression of heparanase in invasive esophageal carcinomas. Jpn J Cancer Res. 2001;92:1062-1073.  [PubMed]  [DOI]
10.  Friedmann Y, Vlodavsky I, Aingorn H, Aviv A, Peretz T, Pecker I, Pappo O. Expression of heparanase in normal, dysplastic, and neoplastic human colonic mucosa and stroma. Evidence for its role in colonic tumorigenesis. Am J Pathol. 2000;157:1167-1175.  [PubMed]  [DOI]
11.  Inoue H, Mimori K, Utsunomiya T, Sadanaga N, Barnard GF, Ueo H, Mori M. Heparanase expression in clinical digestive malignancies. Oncol Rep. 2001;8:539-542.  [PubMed]  [DOI]
12.  El-Assal ON, Yamanoi A, Ono T, Kohno H, Nagasue N. The clinicopathological significance of heparanase and basic fibroblast growth factor expressions in hepatocellular carcinoma. Clin Cancer Res. 2001;7:1299-1305.  [PubMed]  [DOI]
13.  Ikeguchi M, Ueta T, Yamane Y, Hirooka Y, Kaibara N. Quantitative analysis of heparanase messenger RNA expression in hepatocellular carcinoma. J Surg Oncol. 2002;81:148-154; disscusion 154.  [PubMed]  [DOI]
14.  Kim AW, Xu X, Hollinger EF, Gattuso P, Godellas CV, Prinz RA. Human heparanase-1 gene expression in pancreatic adenocarcinoma. J Gastrointest Surg. 2002;6:167-172.  [PubMed]  [DOI]
15.  Koliopanos A, Friess H, Kleeff J, Shi X, Liao Q, Pecker I, Vlodavsky I, Zimmermann A, Büchler MW. Heparanase expression in primary and metastatic pancreatic cancer. Cancer Res. 2001;61:4655-4659.  [PubMed]  [DOI]
16.  Gohji K, Hirano H, Okamoto M, Kitazawa S, Toyoshima M, Dong J, Katsuoka Y, Nakajima M. Expression of three extracellular matrix degradative enzymes in bladder cancer. Int J Cancer. 2001;95:295-301.  [PubMed]  [DOI]
17.  Gohji K, Okamoto M, Kitazawa S, Toyoshima M, Dong J, Katsuoka Y, Nakajima M. Heparanase protein and gene expression in bladder cancer. J Urol. 2001;166:1286-1290.  [PubMed]  [DOI]
18.  Ginath S, Menczer J, Friedmann Y, Aingorn H, Aviv A, Tajima K, Dantes A, Glezerman M, Vlodavsky I, Amsterdam A. Expression of heparanase, Mdm2, and erbB2 in ovarian cancer. Int J Oncol. 2001;18:1133-1144.  [PubMed]  [DOI]
19.  Bitan M, Polliack A, Zecchina G, Nagler A, Friedmann Y, Nadav L, Deutsch V, Pecker I, Eldor A, Vlodavsky I. Heparanase expression in human leukemias is restricted to acute myeloid leukemias. Exp Hematol. 2002;30:34-41.  [PubMed]  [DOI]
20.  Hulett MD, Freeman C, Hamdorf BJ, Baker RT, Harris MJ, Parish CR. Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat Med. 1999;5:803-809.  [PubMed]  [DOI]
21.  Vlodavsky I, Friedmann Y, Elkin M, Aingorn H, Atzmon R, Ishai-Michaeli R, Bitan M, Pappo O, Peretz T, Michal I. Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat Med. 1999;5:793-802.  [PubMed]  [DOI]
22.  Uno F, Fujiwara T, Takata Y, Ohtani S, Katsuda K, Takaoka M, Ohkawa T, Naomoto Y, Nakajima M, Tanaka N. Antisense-mediated suppression of human heparanase gene expression inhibits pleural dissemination of human cancer cells. Cancer Res. 2001;61:7855-7860.  [PubMed]  [DOI]
23.  Demir M, Iqbal O, Hoppensteadt DA, Piccolo P, Ahmad S, Schultz CL, Linhardt RJ, Fareed J. Anticoagulant and antiprotease profiles of a novel natural heparinomimetic mannopentaose phosphate sulfate (PI-88). Clin Appl Thromb Hemost. 2001;7:131-140.  [PubMed]  [DOI]
24.  Miao HQ, Elkin M, Aingorn E, Ishai-Michaeli R, Stein CA, Vlodavsky I. Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate oligodeoxynucleotides. Int J Cancer. 1999;83:424-431.  [PubMed]  [DOI]
25.  Bentolila A, Vlodavsky I, Ishai-Michaeli R, Kovalchuk O, Haloun C, Domb AJ. Poly(N-acryl amino acids): a new class of biologically active polyanions. J Med Chem. 2000;43:2591-2600.  [PubMed]  [DOI]
26.  Yang YJ, Zhang YL, Li X, Dan HL, Lai ZS, Wang JD, Wang QY, Cui HH, Sun Y, Wang YD. Contribution of eIF-4E inhibition to the expression and activity of heparanase in human colon adenocarcinoma cell line: LS-174T. World J Gastroenterol. 2003;9:1707-1712.  [PubMed]  [DOI]
27.  Eccles SA. Heparanase: breaking down barriers in tumors. Nat Med. 1999;5:735-736.  [PubMed]  [DOI]
28.  Tang W, Nakamura Y, Tsujimoto M, Sato M, Wang X, Kurozumi K, Nakahara M, Nakao K, Nakamura M, Mori I. Heparanase: a key enzyme in invasion and metastasis of gastric carcinoma. Mod Pathol. 2002;15:593-598.  [PubMed]  [DOI]
29.  Takaoka M, Naomoto Y, Ohkawa T, Uetsuka H, Shirakawa Y, Uno F, Fujiwara T, Gunduz M, Nagatsuka H, Nakajima M. Heparanase expression correlates with invasion and poor prognosis in gastric cancers. Lab Invest. 2003;83:613-622.  [PubMed]  [DOI]
30.  Chen JP, Lin C, Xu CP, Zhang XY, Wu M. The therapeutic effects of recombinant adenovirus RA538 on human gastric carcinoma cells in vitro and in vivo. World J Gastroenterol. 2000;6:855-860.  [PubMed]  [DOI]
31.  Pan X, Li Z, Zhang M, Wang Y, Pan W, Qi ZT. Therapeutic effect of endostatin-vascular endothelial growth inhibitor recombinant adenoviruses on gastric carcinoma in nude mice. Shijie Huaren Xiaohua Zazhi. 2003;11:1282-1285.  [PubMed]  [DOI]
32.  Li XL, Peng YZ, Yuan ZQ, Tai GP, Chen Y. Preparation of human HSP72 cDNA recombinant adenovirus and its expression in IEC-6 cell. Shijie Huaren Xiaohua Zazhi. 2002;10:1162-1165.  [PubMed]  [DOI]