腺病毒包装

P.O.Box 2345 Beijing 100023, ChinaFax: +86-10-85381893

Email: [email protected] www.wjgnet.comWorld Chin J Digestol 2003 June;11(6):741-744世界华人消化杂志　ISSN 1009-3079 CN 14-1260/R

２００３　年版权归世界胃肠病学杂志社

• 基础研究　BASIC RESEARCH •

内皮抑素－可溶性血管内皮细胞生长抑制因子融合基因重组腺病毒的包装与鉴定

李　　　喆，潘　　　欣，潘　　　卫，曹贵松，闻兆章，方国恩，戚中田，毕建威，华积德

李喆，曹贵松，闻兆章，方国恩，毕建威，华积德，中国人民解放军第二军医大学附属长海医院普通外科　　上海市　　２００４３３

潘欣，潘卫，戚中田，中国人民解放军第二军医大学微生物学教研室上海市　　２００４３３

李喆，男，１９７４－０７－２９生，天津市人，汉族．　１９９８年第二军医大学军医系本科毕业，２０００年第二军医大学博士生．

国家自然科学基金资助项目，　Ｎｏ．３０１７１０５５

项目负责人：潘欣，２００４３３，上海市翔殷路８００号，中国人民解放军第二军医大学微生物学教研室．　　ｐａｎｘｉｎｐｘ＠ｙａｈｏｏ．ｃｏｍ电话：０２１－２５０７０３１４　　　　传真：０２１－２５０７０３１２收稿日期：２００２－０９－１３　　　　接受日期：２００２－１０－０３

Packaging and identification of recom-binant adenovirus carrying endostatin-soluble vascular endothelial growthinhibitor gene

Zhe Li, Xin Pan, Wei Pan, Gui-Song Cao, Zhao-Zhang Wen,Guo-En Fang, Zhong-Tian Qi, Jian-Wei Bi, Ji-De Hua

Zhe Li, Gui-Song Cao, Zhao-Zhang Wen, Guo-En Fang, Jian-Wei Bi,Ji-De Hua,Department of Surgery, Changhai Hospital, Second MilitaryMedical University, Shanghai 200433, China

Xin Pan, Wei Pan, Zhong-Tian Qi, Department of Microbiology, SecondMilitary Medical University, Shanghai 200433, China

Supported by the National Natural Science Foundation of China, No.30171055.

Correspondence to: Dr. Xin Pan, Department of Microbiology, SecondMilitary Medical University, 800 Xiangyin Road, Shanghai 200433,China. [email protected]

Received:2002-09-13 Accepted:2002-10-03

Abstract

AIM:soluble vascular endothelial growth inhibitor gene.

T o acquire recombinant adenovirus carrying endostatin-METHODS:gene (hENDO) and gene of an elastin peptide motif (Val-Pro-IL-3 signal sequence and human endostatinGly-Val-Gly) were amplified with PCR and then ligated withsoluble vascular endothelial growth inhibitor gene (sVEGI).The fusion gene was cloned into adenovirus vector pCA13.The recombinant adenovirus were packaged by means oflipofectamine-mediated gene transfer procedure and identifiedby PCR.

RESULTS:IL-3 signal sequence, entire human endostatin gene, elastinThe fusion gene, about 1 114 bp, which includedpeptide linker gene and soluble vascular endothelial growthinhibitor gene, was successfully cloned into the adenovirusvector pCA13 downstream from the CMV promoter. Themap of restriction enzyme digestion and nuceotide sequencedetermination showed that the fusion gene sequence wasthe same as reported sequence, and in one ORF. Therecombinant adenovirus could be packaged and the titer

of the rough recombinant adenovirus was about 2×TCID 101150/L.

CONCLUSION:gene of hENDO-sVEGI can express the fuion protein inThe recombinant adenovirus containing fusionmammalian cells and secrete to extracellular matrix fromcells. The success of packaging and identification thisrecombinant adenovirus lays the foundation for studyingtumor gene therapy by the fusion gene.

Li Z, Pan X, Pan W, Cao GS, Wen ZZ, Fang GE, Qi ZT, Bi JW, Hua JD.Packaging and identification of recombinant adenovirus carrying endostatin-soluble vascular endothelial growth inhibitor gene. ShijieHuaren Xiaohua Zazhi 2003;11(6):741-744

摘要

目的：构建内皮抑素－可溶性血管内皮细胞生长抑制因子融合基因重组腺病毒载体并包装成重组腺病毒

端引入ＩＬ３信号肽

再与ｓＶＥＧＩ基

因连接

ＰＣＲ法对重组腺病毒进行鉴定．

结果：

构建完成ｈＥＮＤＯ－ｓＶＥＧＩ融合基因重组腺病毒载体

ｈＥＮＤＯ

全长基因总长度约

１　１１４　ｂｐ

插入方向和读码框架均

正确．　可包装出携带融合基因的重组腺病毒

１０１１　　ＴＣＩＤ５０／Ｌ．

结论：

成功构建了ｈＥＮＤＯ－ｓＶＥＧＩ融合基因

为进一步开展肿瘤基因治疗研究奠定了基础．

李喆，潘欣，潘卫，曹贵松，闻兆章，方国恩，戚中田，毕建威，华积德．　内皮抑素－可溶性血管内皮细胞生长抑制因子融合基因重组腺病毒的包装与鉴定．　世界华人消化杂志　　２００３；１１（６）：７４１－７４４

http://www.wjgnet.com/1009-3079/11/741.asp

０　　　引言

目前肿瘤的抗血管生成疗法已成为国内外研究的焦点［１－５］．有２４种血管生成抑制剂正在进行着不同阶段的临床验证．　　由于肿瘤血管生成是一个多步骤的复杂调控过程

742 ISSN 1009-3079 CN 14-1260/R　　　　　　　　　　　　　　　　世界华人消化杂志　　　２００３年６月１５日　　第１１卷　　第６期

不同阶段有不同调控因素发挥主导作用

如

ＩＦＮ

ｍＥｎｄｏｓｔａｔｉｎ和ｍＡｎｇｉｏｓａｔｉｎ联合

基因和融合基因治疗都在动物实验中取得了很好的治疗效果［８，９］　．　我们将两种血管生成抑制剂基因进行融合

达到事半功倍的效果．　我们构建了携

带

ｈＥＮＤＯ－ｓＶＥＧＩ融合基因的重组腺病毒载体

ｐＪＭ１７

质粒

内皮抑素和可溶性血

管内皮细胞生长抑制因子基因的克隆及鉴定由微生物

教研室完成

为微生物教研室

保存．　Ｔａｑ　ＤＮＡ

多聚酶

　ｍａｒｋｅｒ均购于华美

生物工程公司

ＰＣＲ引物由上海生工公司合成：引物１为　

５

；引物２为

５

引物３为

５

引物４为

５

以ｈＥＮＤＯ基因为模板用引物

１和引物２扩增ＩＬ３／ｈＥＮＤＯ－ｌｉｎｋｅｒ

片段

酶切位点和ＩＬ３

信号肽序列

酶切位点

在５

酶切位点

酶切位点．

ＰＣＲ反应进程为变性

５　ｍｉｎ

５０　共循环３０

次

　延伸

１０　ｍｉｎ．　产物经纯化后

和Ｂａｍ

Ｈ

将ＩＬ３／ｈＥＮＤＯ－ｌｉｎｋｅｒ片段从携带目的基因

的

ｐＭＤ１８－Ｔ　载体上游离下来

／Ｂａｍ

Ｈ

－Ｅｃｏ

Ｒ

ｓＶＥＧＩ　片段进行连接

钙化菌

　以Ｈｉｎ

ｄ

＋Ｅｃｏ

Ｒ

酶切鉴定

阳性克隆或Ｓａ

ｃ

　用ｌｉｐｏｆｅｃｔＡＭＩＮＥ共转染２４孔板中（用板中间的孔）

的２９３细胞．　约

４　ｄ

－７０－３７　

制备Ａｄ粗提液（混合

克隆）

保存．　包装好的腺病毒命名为：　ＡｄＣＡ１３－ｈＥＮＤＯ－ｓＶＥＧＩ．　　收集２９３

细胞

接种９６孔板每孔细胞悬

液１００　ｕＬ．　第２

天当细胞铺满时

－７０－３７　

　每孔加

０．１　ｍＬ纵行＃１１和１２用做阴性对照

５　％　ＣＯ２孵箱培养１０　ｄ

ｓ的

统计方法计算病毒滴度．

２　　　结果

２．１融合基因腺病毒载体的构建和酶切鉴定　　扩增的ＩＬ３／ｈＥＮＤＯ－ｌｉｎｋｅｒ片段约为

６４７　ｂｐ

然后将目的基因取下＋Ｂａｍ

Ｈ

＋　Ｂａｍ

Ｈ

证明融合基因被

正确克隆到ｐＣＡ１３载体　（图３）．　Ｂｇ

ｌ

564 bp

1 2 3

ＤＮＡ／　

ＥｏｃＲ

李喆，　等．　内皮抑素－可溶性血管内皮细胞生长抑制因子融合基因重组腺病毒的包装与鉴定 743

Hin

+Eco R ＩHin

Sal Hin

Ｉ+Bam H Ｉ

pCA13

SV40polyA

6 950 bp

Sac Ｉ

Hin

Ampr

图２　　ｐＣＡ１３　－ｈＥＮＤＯ－ｓＶＥＧＩ质粒的构建流程示意图．

1 2 3 4

１：　ｍａｒｋｅｒ（２１　２２７　ｂｐ，５　１４８　ｂｐ，４　２６９　ｂｐ，３５３０　ｂｐ，２　０２７　ｂｐ，１　９０４　ｂｐ，１　５８７　ｂｐ，１　３７５　ｂｐ，９４１　ｂｐ，８３１　ｂｐ，５６４　ｂｐ）２：Ｄｉｇｅｓｔｉｎｇ　ｗｉｔｈ　Ｈｉｎ

ｄ

　ａｎｄ　　Ｅｃｏ

Ｒ

　（４７６　ｂｐ，　７．６９　ｋｂ）．

图３　ｐＣＡ１３　－ｈＥＮＤＯ－ｓＶＥＧＩ质粒的目的基因酶切鉴定

1 2 3 4

１：　ｍａｒｋｅｒ　（２１　２２７　ｂｐ，５　１４８　ｂｐ，　４　２６９　ｂｐ，３５３０　ｂｐ，２　０２７　ｂｐ，１　９０４　ｂｐ，１　５８７　ｂｐ，１　３７５　ｂｐ，９４１　ｂｐ，８３１　ｂｐ，５６４　ｂｐ）２：　Ｄｉｇｅｓｔｉｎｇ　ｗｉｔｈ　Ｈｉｎ

ｄ

　（１　７０７　ｂｐ，６．３　ｋｂ）

４：　Ｄｉｇｅｓｔｉｎｇ　ｗｉｔｈ　Ｓａ

ｃ

约４　ｄ　可见ＣＰＥ出现．　收集细胞及上

清

反复冻融

３

次

　１０　ｍｉｎ　扩增出

ｓＶＥＧＩ

片段

４８－７２　ｈ收集呈

ＣＰＥ

表现的细胞

制备Ａｄ　ｈＥＮＤＯ－ｓＶＥＧＩ

粗提液

１０

１１

　ＴＣＩＤ５０／Ｌ．

３　　　讨论

抗血管生成疗法作为一种新兴疗法

将其融合并成功构建携带融合基

因的重组腺病毒载体

既对血管生成产生协同抑制作用

免疫调节作用杀伤肿

瘤细胞

没有耐药性

未见毒副作用

［２０，２１］

期临床试验

．　　内皮抑素基因是一个很好的治疗基因

［２２－３１］

．

ＶＥＧＩ是从人脐静脉内皮细胞ｃＤＮＡ文库中筛选得到的一个ＴＮＦ家族的新成员［３２］．　ＶＥＧＩ

属于

体外血管生成模型中显著抑制

牛主动脉内皮细胞在胶原纤维中形成管样结构

［３３］

抑制肿瘤生

长

发现异种

移植的肿瘤生长明显受到抑制．　以上结果说明分泌到肿

瘤局部的

ＶＥＧＩ

通过抑制新生血管生成抑制肿瘤生长

．

除抑制内皮细胞增生外

Ｈｅｌａ

细胞

加入合成抑制剂环已酮亚胺

时

但并不妨碍对其进行研究利用

．　我

们参照

Ｉｎｖｉｖｏｇｅｎ

基因公司的作法

保证翻译

后两蛋白空间构象不受影响

744 ISSN 1009-3079 CN 14-1260/R　　　　　　　　　　　　　　　　世界华人消化杂志　　　２００３年６月１５日　　第１１卷　　第６期

and opportunities with novel human therapies. J Pathol 2000;190:330-337

5Vacca A, Ribatti D, Pellegrino A, Dammacco F. Angiogenesis andanti-angiogenesis in human neoplasms. Recent developments andthe therapeutic prospects. Ann Ital Med Int 2000;15:7-19

Parangi S, O'Reilly M, Christofori G, Holmgren L, Grosfeld J,Folkman J, Hanahan D. Antiangiogenic therapy of transgenicmice impairs de-novo tumor-growth. Proc Natl Acad Sci USA1996;93:2002-2007

7Boehm T, Folkman J, Browder T, O'Reilly MS. Anti-angiogenictherapy of experimental cancer does not induce acquired drugresistance. Nature 1997;390:404-407

Scappaticci FA, Smith R, Pathak A, Schloss D, Lum B, Cao Y,Johnson F, Engleman EG, Nolan GP. Combination angiostatinand endostatin gene transfer induces synergistic antiangiogenicactivity in vitro and antitumor efficacy in leukemia and solidtumors in mice. Mol Ther 2001;3:186-196

Scappaticci FA, Contreras A, Smith R, Bonhoure L, Lum B,Cao Y, Engleman EG, Nolan GP. Statin-AE: a novel angiostatin-endostatin fusion protein with enhanced antiangiogenic andantitumor activity. Angiogenesis 2001;4:263-268

10Folkman J. Antiangiogenic gene therapy. Proc Natl Acad Sci USA1998;95:9064-9066

11Tanaka T, Cao Y, Folkman J, Fine HA. Viral vector-targetedantiangiogenic gene therapy utilizing an angiostatin comple-mentary DNA. Cancer Res 1998;58:3362-3369

Cao Y, O'Reilly MS, Marshall B, Flynn E, Ji RW, Folkman J.Expression of angiostatin cDNA in a murine fibrosarcoma sup-presses primary tumor growth and produces long-term dor-mancy of metastases. J Clin Invest 1998;101:1055-1063

13Nguyen JT, Wu P, Clouse ME, Hlatky L, Terwilliger EF. Adeno-associated virus-mediated delivery of antiangiogenic factorsas an antitumor strategy. Cancer Res 1998;58:5673-5677

Machein MR, Risau W, Plate KH. Antiangiogenic gene therapyin a rat glioma model using a dominant-negative vascular en-dothelial growth factor receptor2. Hum Gene Ther 1999;10:1117-1128

Indraccolo S, Minuzzo S, Gola E, Habeler W, Carrozzino F,Noonan D, Albini A, Santi L, Amadori A, Chieco-Bianchi L.Generation of expression plasmids for angiostatin, endostatinand TIMP-2 for cancer gene therapy. Int J Biol Markers 1999;14:251-256

16Chen QR, Kumar D, Stass SA, Mixson AJ. Liposomes complexedto plasmids encoding angiostatin and endostatin inhibit breastcancer in nude mice. Cancer Res 1999;59:3308-3312

Matsuda KM, Madoiwa S, Hasumi Y, Kanazawa T, Saga Y,Kume A, Mano H, Ozawa K, Matsuda M. A novel strategy forthe tumor angiogenesis-targeted gene therapy :generation ofangiostatin from endogenous plasminogen by protease genetransfer. Cancer Gene Ther 2000;7:589-596

18Bergers G, Hanahan D. Cell factories for fighting cancer. Nat Biotechnol 2001;19:20-21

Regulier E, Paul S, Marigliano M, Kintz J, Poitevin Y, LedouxC, Roecklin D, Cauet G, Calenda V, Homann HE. Adenovirus-mediated delivery of antiangiogenic genes as an antitumorapproach. Cancer Gene Ther 2001;8:45-54

Berger AC, Feldman AL, Gnant MF, Kruger EA, Sim BK, HewittS, Figg WD, Alexander HR, Libutti SK. The angiogenesis inhibitor, endostatin, does not affect murine cutaneous woundhealing. J Surg Res 2000;91:26-31

Bloch W, Huggel K, Sasaki T, Grose R, Bugnon P, Addicks K,Timpl R, Werner S. The angiogenesis inhibitor endostatin im-pairs blood vessel maturation during wound healing. FASEB J2000;14:2373-2376

Yoon SS, Eto H, Lin CM, Nakamura H, Pawlik TM, Song SU,

Tanabe KK. Mouse endostatin inhibits the formation of lungand liver metastases. Cancer Res 1999;59:6251-6256

23Szary J, Szala S. Intra-tumoral administration of naked plas-mid DNA encoding mouse endostatin inhibits renal carcinomagrowth. Int J Cancer 2001;91:835-839

Ding I, Sun JZ, Fenton B, Liu WM, Kimsely P, Okunieff P, MinW. Intratumoral administration of endostatin plasmid inhibitsvascular growth and perfusion in MCa-4 murine mammarycarcinomas. Cancer Res 2001;61:526-531

Blezinger P, Wang J, Gondo M, Quezada A, Mehrens D, FrenchM, Singhal A, Sullivan S, Rolland A, Ralston R, Min W. Sys-temic inhibition of tumor growth and tumor metastases byintramuscular administration of the endostatin gene. Nat Biotechnol 1999;17:343-348

Sauter BV, Martinet O, Zhang WJ, Mandeli J, Woo SL. Aden-ovirus-mediated gene transfer of endostatin in vivo results inhigh level of transgene expression and inhibition of tumor growthand metastases. Proc Natl Acad Sci USA 2000;97:4802-480727

Read TA, Sorensen DR, Mahesparan R, Enger PO, Timpl R,Olsen BR, Hjelstuen MH, Haraldseth O, Bjerkvig R. Localendostatin treatment of gliomas administered by microencap-sulated producer cells. Nat Biotechnol 2001;19:29-34

Joki T, Machluf M, Atala A, Zhu J, Seyfried NT, Dunn IF, AbeT, Carroll RS, Black PM. Continuous release of endostatin frommicroencapsulated engineered cells for tumor therapy. Nat Biotechnol 2001;19:35-39

Feldman AL, Restifo NP, Alexander HR, Bartlett DL, Hwu P,Seth P, Libutti SK. Antiangiogenic gene therapy of cancer uti-lizing a recombinant adenovirus to elevate systemic endostatinlevels in mice. Cancer Res 2000;60:1503-1506

Chen CT, Lin J, Li Q, Phipps SS, Jakubczak JL, Stewart DA,Skripchenko Y, Forry-Schaudies S, Wood J, Schnell C, HallenbeckPL. Antiangiogenic gene therapy for cancer via systemic ad-ministration of adenoviral vectors expressing secretable endostatin. Hum Gene Ther 2000;11:1983-1996

Wen XY, Bai Y, Stewart AK. Adenovirus-mediated humanendostatin gene delivery demonstrates strain-specific antitu-mor activity and acute dose-dependent toxicity in mice. Hum Gene Ther 2001;12:347-358

Tan KB, Harrop J, Reddy M, Young P, Terrstt J, Emery J,Moore G,Truneh A. Characterization of a novel TNF-like ligand and re-cently described TNF ligand and TNF receptor superfamily genesand their constitutive and inducible expression in hematopoieticand non-hematopoietic cells. Gene 1997;204:35-46

Yue TL, Ni J, Romanic AM, Gu JL, Keller P, Wang C, Kumar S,Yu GL, Hart TK, Wang XK, Xia ZG, DeWolf WE Jr, FeuersteinGZ. TL1,a novel tumor necrosis factor-like cytokine,inducesapoptosis in endothelial cells. Involvement of activation of stressprotein kinases (stress-activated protein kinase and p38 mito-gen-activated protein kinase) and caspase-3-like protease. JBiol Chem 1999;274:1479-1486

Zhai Y, Yu J, Iruela AL, Huang WQ, Wang Z, Hayes AJ, Lu J,Jiang G, Rojas L, Lippman ME, Ni J, Yu GL, Li LY. Inhibition ofangiogenesis and breast cancer xenograft tumor growth byVEGI,a novel cytokine of the TNF superfamily. Int J Cancer1999;82:131-136

Zhai Y, Ni J, Jiang GW, Lu JM, Xing L, Lincoln C, Carter KC,Janat F, Kozak D, Xu SM, Rojas L, Aggarwal BB, Ruben S, LiLY, Gentz R, Yu GL. VEGI, a novel cytokine of the tumor necro-sis factor family, is an angiogenesis inhibitor that suppresses thegrowth of colon carcinomas in vivo. FASEB J 1999;13:181-18936

Harids V, Shrivastava A, Su J, Yu GL, Ni J, Liu D, Chen SF, Ni Y,Ruben SM, Gentz R, Aggarwal BB. VEGI,a new member of the TNFfamily activates nuclear factor-KappaB and c-jun n-terminal kinaseand modulates cell growth. Oncogene 1999;18:6496-6504