基础研究 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-06-15; 11(6): 741-744
在线出版日期: 2003-06-15. doi: 10.11569/wcjd.v11.i6.741
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
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.
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
基金项目: 国家自然科学基金资助项目, No. 30171055.
通讯作者: 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
修回日期: 2002-09-20
接受日期: 2002-10-03
在线出版日期: 2003-06-15

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, 为下一步基因治疗打下基础.

方法

用PCR方法在hENDO基因的5'端引入IL3信号肽、3'端引入弹性蛋白连接肽序列(linker), 再与sVEGI基因连接, 插入到pCA13腺病毒载体中. 用脂质体介导包装出重组腺病毒, 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, 融合基因包括IL3信号肽、hENDO全长基因、弹性蛋白连接肽序列和sVEGI基因, 总长度约1114 bp, 经酶切鉴定、序列分析证实克隆序列、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, 用TCID50法测定粗制重组腺病毒滴度为2×1011 TCID50/L.

结论

成功构建了hENDO-sVEGI融合基因, 连接肽序列使两蛋白空间构象不受影响, IL3信号肽保证蛋白可被分泌至胞外. 完成携带融合基因的重组腺病毒的包装与鉴定, 为进一步开展肿瘤基因治疗研究奠定了基础.

关键词: N/A
引文著录: 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
Packaging and identification of recombinant adenovirus carrying endostatin-soluble vascular endothelial growth inhibitor 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 Military Medical University, Shanghai 200433, China
Xin Pan, Wei Pan, Zhong-Tian Qi, Department of Microbiology, Second Military Medical University, Shanghai 200433, China
Supported by: the National Natural Science Foundation of China, No. 30171055.
Correspondence to: Dr. Xin Pan, Department of Microbiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. panxinpx@yahoo.com
Received: September 13, 2002
Revised: September 20, 2002
Accepted: October 3, 2002
Published online: June 15, 2003

AIM

To acquire recombinant adenovirus carrying endostatin-soluble vascular endothelial growth inhibitor gene.

METHOD

IL-3 signal sequence and human endostatin gene (hENDO) and gene of an elastin peptide motif (Val-Pro-Gly-Val-Gly) were amplified with PCR and then ligated with soluble vascular endothelial growth inhibitor gene (sVEGI). The fusion gene was cloned into adenovirus vector pCA13. The recombinant adenovirus were packaged by means of lipofectamine-mediated gene transfer procedure and identified by PCR.

RESULTS

The fusion gene, about 1114 bp, which included IL-3 signal sequence, entire human endostatin gene, elastin peptide linker gene and soluble vascular endothelial growth inhibitor gene, was successfully cloned into the adenovirus vector pCA13 downstream from the CMV promoter. The map of restriction enzyme digestion and nuceotide sequence determination showed that the fusion gene sequence was the same as reported sequence, and in one ORF. The recombinant adenovirus could be packaged and the titer of the rough recombinant adenovirus was about 2×1011 TCID50/L.

CONCLUSION

The recombinant adenovirus containing fusion gene of hENDO-sVEGI can express the fuion protein in mammalian cells and secrete to extracellular matrix from cells. The success of packaging and identification this recombinant adenovirus lays the foundation for studying tumor gene therapy by the fusion gene.

Key Words: N/A
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, 不同阶段有不同调控因素发挥主导作用, 因而单一针对某一治疗环节的药物疗效有限. 不同机制的血管生成抑制剂有协同治疗作用, 如IFNα和TNP470[6]. 联合应用鼠内皮抑素(mEndostatin)和血管抑素(mAngiosatin)能彻底根除小鼠体内的移植瘤[7], 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, 表达出具有两种药物活性的融合蛋白, 从不同环节阻断肿瘤血管生成, 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, 以期为肿瘤的抗血管生成基因治疗提供实验基础.

1 材料和方法
1.1 材料

pCA13、pJM17质粒、293细胞由第二军医大学微生物教研室保存并提供; 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; PCR产物克隆载体pMD-18-T为大连TaKaRa公司产品; 大肠杆菌DH5α为微生物教研室保存. Taq DNA多聚酶、各种限制性内切酶、T4DNA连接酶、λDNA/ EcoR I +Hind III marker均购于华美生物工程公司; DMEM, lipofectAMINETM为Gibco产品; PCR引物由上海生工公司合成: 引物1为5'-GCAAGCTTGCCACCATGAGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCC

GGACTCCAACACAGCCACCGCGAC-3'; 引物2为5'-CACCGAATTCGTACCTACTCCTGGTACCTTGGAGGCAGTCATGAAG-3'; 引物3为5'-ACGAATTCGGTGCCAGTTGTGAGAC-3'; 引物4为5' -GCGGATCCCTATAGTAAGAAGGCTCC-3'.

1.2 方法

用PCR方法, 以hENDO基因为模板用引物1和引物2扩增IL3/hENDO-linker片段, 在5'端引入HindⅢ酶切位点和IL3信号肽序列, 3'端引入弹性蛋白连接肽序列和EcoRⅠ酶切位点; 以sVEGI基因为模板用引物3'和引物4扩增sVEGI片段, 在5'端引入EcoRⅠ酶切位点, 3'端引BamHⅠ酶切位点. PCR反应进程为: 94 °C变性5 min; 94 °C 45 s, 50 °C 45 s, 72 °C 120 s, 共循环30次; 最后, 72 °C 延伸10 min. 产物经纯化后, 分别克隆到pMD-18-T载体, 送上海生工公司用M13引物进行DNA自动测序. 腺病毒载体pCA13用HindⅢ和BamHⅠ双酶切线性化, 用HindⅢ/EcoRⅠ将IL3/hENDO-linker片段从携带目的基因的pMD18-T 载体上游离下来, 用EcoRⅠ/BamHⅠ将sVEGI片段从携带目的基因的pMD18-T 载体上游离下来, 在T4DNA连接酶作用下, 将线性化的pCA13与HindⅢ-EcoRⅠIL3/hENDO-linker片段和EcoRⅠ- BamHⅠsVEGI 片段进行连接, 转化DH5α钙化菌, 分别挑取克隆进行质粒的小量制备; 以HindⅢ+ BamHⅠ, HindⅢ+EcoRⅠ, EcoRⅠ+ BamHⅠ酶切鉴定阳性克隆, 分别以载体上的BglⅡ或SacⅠ位点酶切鉴定融合基因被正确克隆在CMV启动子的下游以确认读框正确. 将质粒pJM17与重组腺病毒载体以1:5比例混合, 用lipofectAMINE共转染24孔板中(用板中间的孔)的293细胞. 约4 d, 可见细胞病变(CPE)出现. 收集细胞及上清, -70-37 °C反复冻融3次, 用上清液(含Ad)50 μL加完全培养液200 μL感染293细胞, 约2 d (48 h) 收集呈CPE表现的细胞及上清, 制备Ad粗提液(混合克隆), 同法大量扩增病毒, -70 °C保存. 包装好的腺病毒命名为: AdCA13-hENDO-sVEGI. 收集293细胞, 计数, 调整细胞浓度为108/L, 接种96孔板每孔细胞悬液100 uL. 第2天当细胞铺满时, 吸去上清, 加入各稀释度的粗制重组腺病毒. 混合收集到的Ad粗提液10 mL, -70-37 °C反复冻融3次, 4000 r/min 10 min, 用完全培养液将病毒作连续10倍稀释, 从10-1-10-10作为滴定的稀释度. 用最后8个稀释液加入96孔板, 每孔加0.1 mL, 每个稀释度10孔; 纵行#11和12用做阴性对照, 每孔加0.1 mL 完全培养液, 5% CO2孵箱培养10 d, 倒置显微镜读板, 确定每行阳性孔的比率. 按Karber'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, sVEGI片段为476 bp (图1). 将PCR扩增的目的基因分别直接亚克隆于pMD18-T载体上, 然后将目的基因取下, 与经相应酶切的pCA13载体连接构建携带融合基因的腺病毒载体(图2). 以HindⅢ+BamHⅠ, HindⅢ+EcoRⅠ, EcoRⅠ+ BamHⅠ酶切pCA13-hENDO-sVEGI 分别得到1 114 bp hENDO-sVEGI片段, 647 bp IL3/hENDO-linker片段和467 bp的sVEGI片段, 证明融合基因被正确克隆到pCA13载体 (图3). BglⅡ、SacⅠ位点酶切鉴定融合基因被正确克隆在CMV启动子的下游以确认读框正确(图4).

图1
图1 IL3/hENDO-linker和sVEGI基因PCR扩增电泳图. 1: λDNA/ EocRⅠ+HindⅢ marker(21227 bp, 5148 bp, 4269 bp, 3530 bp, 2027 bp, 1904 bp, 1587 bp, 1375 bp, 941 bp, 831 bp, 564 bp); 2: 647 bp的IL3/hENDO-linker片段; 3: 476 bp的VEGI片段.
图2
图2 pCA13 -hENDO-sVEGI质粒的构建流程示意图. 1: λDNA/ EcoRⅠ+HindⅢ marker(21 227 bp, 5 148 bp, 4 269 bp, 3 530 bp, 2 027 bp, 1 904 bp, 1 587 bp, 1 375 bp, 941 bp, 831 bp, 564 bp); 2: Digesting with HindⅢ and BamHⅠ(1 114 bp, 6.95 kb); 3: Digesting with HindⅢ and EcoRⅠ (647 bp, 7.49 kb); 4: Digesting with EcoRⅠand BamHⅠ(476 bp, 7.69 kb).
图3
图3 pCA13 -hENDO-sVEGI质粒的目的基因酶切鉴定. 1: λDNA/ EcoRⅠ+HindⅢ marker (21227 bp, 5148 bp, 4269 bp, 3530 bp, 2027 bp, 1904 bp, 1587 bp, 1375 bp, 941 bp, 831 bp, 564 bp); 2: Digesting with HindⅢ and BamHⅠ(1114 bp, 6.95 kb); 3: Digesting with BglⅡ(1707 bp, 6.3 kb); 4: Digesting with SacⅠ(1535 bp, 2.0 kb, 4.5 kb).
图4
图4 pCA13 -hENDO-sVEGI质粒目的基因插入部位的酶切鉴定.
2.2 携带融合基因的重组腺病毒

质粒pCA13-hENDO-sVEGI与pJM17用lipofectAMINETM共转染293细胞进行同源重组, 约4 d 可见CPE出现. 收集细胞及上清, -70-37 °C反复冻融3次, 4000 r/min, 4 °C 10 min, 用引物3和引物4对病毒裂解液行PCR, 扩增出sVEGI片段, 说明重组腺病毒包装成功. 用上清液(含Ad)50 μL加完全培养液200 μL接种293细胞, 48-72 h收集呈CPE表现的细胞, 制备Ad粗提液(混合克隆). 同法大量扩增病毒, 制备Ad hENDO-sVEGI粗提液, 并进行效价滴定, 病毒滴度为2×1011 TCID50/L.

3 讨论

抗血管生成疗法作为一种新兴疗法, 还存在不少缺点有待改进. 内源性血管生成抑制剂的基因治疗被认为是最有前途的治疗策略[10-19]. 我们选择两个强效的内源性血管生成抑制基因, 将其融合并成功构建携带融合基因的重组腺病毒载体, 期望融合蛋白分子能发挥两种分子的活性, 既对血管生成产生协同抑制作用, 又通过sVEGI的直接细胞毒、诱导凋亡、免疫调节作用杀伤肿瘤细胞, 产生最理想的协同抑瘤效应.

内皮抑素是迄今所发现的最有效的血管生成抑制剂之一, 没有耐药性, 不需进一步给药就能延长肿瘤的休眠期[7], 未见毒副作用[20,21], 在美国已进入了Ⅱ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Ⅱ型跨膜蛋白, 基因定位于人染色体9q32, 在部分内皮细胞中大量表达, 其氨基酸序列与肿瘤坏死因子(TNF)家族成员有20-30 %的同源性. 其可溶性的胞外分子(sVEGI)能特异性显著抑制牛肺动脉内皮细胞(BPAEC)和人脐静脉内皮细胞(HUVEC)增生[33], 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], 体内鸡胚尿囊膜新生血管实验中可剂量依赖性抑制FGF或VEGF诱导的毛细血管生成. 基因转移可溶性分子可降低鼠源性MC-38结肠癌细胞的成瘤性, 抑制肿瘤生长; 组织学检测发现肿瘤中的微血管密度明显降低, 内皮细胞增生受抑显著, 凋亡数量增加[34]. Zhai et al [35]将表达可溶性VEGI的中国仓鼠卵巢(CHO)细胞与人乳腺癌细胞MDA-MB-231混合注射裸鼠体内, 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, Harids et al [36]发现sVEGI对乳癌细胞MCF-7、Hela细胞、髓样细胞U-937和ML-la四种肿瘤细胞均有抑制作用, 加入合成抑制剂环已酮亚胺时, 细胞毒作用更加明显, 因此推测是通过抑制蛋白合成而发挥作用的.

内皮抑素与VEGI这两种血管生成抑制因子的作用机制目前尚不明确, 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, 两基因之间采用缬氨酸-脯氨酸-甘氨酸-缬氨酸-甘氨酸(Val-Pro-Gly-Val-Gly)五个氨基酸的弹性蛋白酶连接肽, 保证翻译后两蛋白空间构象不受影响, IL3信号肽保证蛋白可被分泌至胞外.

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