SV40T抗原胃壁细胞特异性表达载体的构建与鉴定

摘要

目的: 构建在胃壁细胞中特异性表达SV40T抗原的真核表达载体并进行鉴定.

方法: 采用酚-氯仿法从昆明小鼠肝细胞中提取基因组DNA, 聚合酶链式反应(PCR)扩增H⁺/K⁺ATPase β亚基启动子, 产物命名为HK. 将PCR产物纯化回收后与pMT18-T载体相连, 并将其克隆至真核表达载体pcDNA3.1(-), 构建pcDNA3.1(-)/HK; 从含SV40T基因片段的质粒pLITAg中酶切回收SV40T基因, 与pcDNA3.1(-)/HK相连, 构建胃壁细胞特异性表达载体pcDNA3.1(-)/HKSV, 并测序鉴定.

结果: pcDNA3.1(-)/HKSV用XbaⅠ、BamHⅠ双酶切可得到1 kb H⁺/K⁺ATPase β亚基启动子, 2.7 kb SV40T基因与5.4 kb pcDNA3.1(-)载体3条DNA条带. 用XbaⅠ、KpnⅠ双酶切电泳, 可见到约3.7与5.4 kb的两条DNA条带; 用BamHⅠ单酶切电泳, 可见到2.7与6.4 kb的2条DNA条带; 用EcoR Ⅰ单酶切, 只见到约9.1 kb的1条DNA条带, 酶切电泳结果均与设计一致. 测序结果显示, H⁺/K⁺ATPase β亚基启动子与SV40T基因成功构建于pcDNA3.1(-)真核表达载体中.

结论: 构建在胃壁细胞中特异性表达SV40T基因的真核表达载体, 为进一步转基因小鼠及胃癌动物模型的建立提供了稳定、可靠的分子工具.

关键词: SV40病毒; T 抗原; 真核表达; 载体构建

Construction and identification of a specific expression vector for SV40 large T antigen in gastric parietal cell

Hui Chen, Yi-Fang Hou, Xiao-Ping Le, Hui Jin, Hui-Jie Ma, Qin-Xian Zhang

Hui Chen, Department of Cell Biology and Medical Genetics, College of Basic Medicine Sciences of Zhengzhou University; He'nan Provincial Key Laboratory of Molecular Medicine, Zhengzhou 450052, He'nan Province, China

Xiao-Ping Le, Hui Jin, Hui-Jie Ma, Qin-Xian Zhang, Department of Histology and Embryology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450052, He'nan Province, China

Yi-Fang Hou, Department of Obstetrics and Gynecology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, He'nan Province, China

Supported by: He'nan Provincial Key Technologies R & D Program, No. 0623031400.

Correspondence to: Dr. Qin-Xian Zhang, Department of Histology and Embryology, College of Basic Medical Sciences of Zhengzhou University, Zhengzhou 450052, He'nan Province, China. qxz53@zzu.edu.cn

Received: January 11, 2007
Revised: March 1, 2007
Accepted: March 23, 2007
Published online: June 28, 2007

Abstract

AIM: To construct and identify a eukaryotic specific expression vector of SV40 large T antigen in gastric parietal cell.

METHODS: Genome DNA was extracted from liver cells of Kunming mice by a phenol-chloroform method. The H⁺/K⁺ ATPase β subunit promoter gene was amplified by polymerase chain reaction (PCR) and the product was named as HK. This was then ligated with the pMT18-T vector after purification. A cloning vector of pcDNA3.1 (-)/HK was constructed by ligating the H⁺/K⁺ATPase β subunit promoter and the eukaryotic vector pcDNA3.1 (-). SV40 large T genes were digested by restricted enzymes from PLITAg recombinant and then inserted into the prokaryotic expression vector pcDNA3.1 (-)/HK. Thus, an expression vector specific for pcDNA3.1 (-)/HKSV was constructed and identified in gastric parietal cell.

RESULTS: Three DNA bands were seen when pcDNA3.1(-)/HKSV was digested by XbaⅠand BamHⅠ, which were the 1 kb H+/K+ ATPase β subunit promoter gene, the 2.7 kb SV40T gene and the 5.4 kb pcDNA3.1(-) vector. Two DNA bands, 3.7 kb and 5.4 kb were seen when pcDNA3.1(-)/HKSV was digested by XbaⅠ and KpnⅠ. Another two DNA bands, 2.7 kb and 6.4 kb, were seen when pcDNA3.1(-)/HKSV was digested by BamHⅠ. One DNA band, 9.1 kb, was seen when pcDNA3.1(-)/HKSV was digested by EcoRⅠ. All electrophoresis results were consistent with the design. The sequencing results showed that H⁺/K⁺ ATPase β subunit promoter and SV40 large T gene had been successfully cloned into pcDNA3.1(-) eukaryotic vector.

CONCLUSION: The recombinant plasmid pcDNA3.1 (-)/HKSV which is specially expressed in gastric parietal cell, is a stable and valuable molecular tool for establishing transgenic mice and animal models of gastric carcinoma.

Key Words: SV40 virus; T antigen; Eukaryotic expression; Vector construction

0 引言

SV40(simian virus 40)是1960年代初发现分离的猿猴肾细胞病毒, 属乳多空病毒科多型瘤病毒属中的一种小DNA肿瘤病毒, 具有转化动物细胞和诱发肿瘤的特性. SV40病毒的细胞转化特性主要是与早期蛋白T抗原有关, 国外诸多研究证实了SV40T转基因小鼠模型的发瘤率很高, 可用于肿瘤发病机制的研究与转基因动物模型的建立, 如利用SV40T基因已建立了小鼠乳腺癌、脑脉络丛乳头状瘤、胰腺癌等转基因动物模型. 胃壁细胞是胃底腺的主要细胞类型之一, 能合成和分泌盐酸, 从而刺激胃肠道内分泌细胞和胰液的分泌. H⁺/K⁺ATP酶基因在胃壁细胞中特异性表达, 和胃酸的合成与分泌有着直接的关系. Gordon建立了在胃壁细胞中特异性表达人生长因子(hGH)、内在因子(INF)的转基因动物模型, 研究了胃黏膜上皮细胞的发生、演化过程, 构建的HKATP/SV40T胃癌转基因小鼠动物模型, 为胃癌的诊断、治疗提供了十分有用的实验材料. 但此方面的研究在国内未见报道, 我们拟构建在胃壁细胞中特异性表达SV40T基因的真核表达载体, 为转基因小鼠及胃癌动物模型的建立提供稳定、可靠的分子工具.

1 材料和方法

1.1 材料

昆明小鼠由河南省实验动物中心提供, PLITAg由美国Jeffrey I. Gordon 惠赠, 克隆载体pcDNA3.1(-)由中南大学生殖与干细胞工程研究所刘永波博士惠赠, 载体pMD18-T由郑州大学公共卫生学院宋春花博士惠赠. 大肠杆菌DH5α为本室保存. T4 DNA连接酶、DL15000 DNA Marker、限制性内切酶BamHⅠ、EcoR Ⅰ、KpnⅠ和XbaⅠ为TaKaRa公司产品; 蛋白酶K、RNA酶A购自Sigma公司; 胰蛋白胨、酵母提取物购自Oxoid公司; 凝胶回收试剂盒购自TaKaRa公司.

1.2 方法

采用酚-氯仿法从昆明小鼠肝细胞中提取基因组DNA, 紫外分光光度计测定DNA的浓度和纯度, 置-20℃保存备用.

1.2.1 PCR扩增: 以小鼠基因组DNA为模板, 设计引物扩增H⁺/K⁺ATPase b亚基启动子片段, P1: 5'-TCTAGAGCTCTTTCCTCTGGGTC-3'(含XbaⅠ位点), P2: 5'-CTCGGATTCGTCCTCTCCTGCTT-3'(含BamHⅠ位点). 反应条件为: 94℃预变性3 min; 94℃ 45 s, 60℃ 45 s, 72℃ 60 s, 共35个循环, 最后72℃延伸10 min, PCR产物命名为HK. 将PCR产物纯化回收后与pMT18-T载体相连, 转化感受态E.coli DH5α细胞, 从转化平板上随机挑取8个单菌落, 37℃摇菌过夜培养, 取菌液提取质粒DNA, XbaⅠ、BamHⅠ双酶切鉴定出阳性克隆, 命名为pMT/HK, 送北京三博远志生物工程公司测序.

1.2.2 构建pcDNA3.1/HK质粒: 将pMT/HK用XbaⅠ、BamHⅠ双酶切, 琼脂糖凝胶电泳, 切胶回收约1060 bp的DNA片段, 将回收产物与用XbaⅠ、BamHⅠ双酶切的pcDNA3.1(-)质粒连接, 目的片段与载体分子摩尔比为3:1, 45℃水浴5 min, 16℃连接16 h后, 按常规方法转化感受态DH5α细胞, 从转化平板上任意挑单菌落, 提取质粒DNA, 限制性内切酶消化鉴定出阳性重组克隆, 记作pcDNA3.1/HK.

1.2.3 构建pcDNA3.1/HKSV质粒: 将PLITAg(含SV40T基因片段)质粒用BamHⅠ酶切, 琼脂糖凝胶电泳, 切胶回收约2700 bp的DNA片段, 将回收产物与用BamHⅠ酶切的pcDNA3.1/HK连接, 目的片段与载体分子摩尔比为3:1, 反应体积为10 mL, 连接反应体系16℃连接过夜, 取2 mL连接产物转化至E.coli DH5α细胞中, 从转化平板上挑取单菌落, 37℃摇菌过夜培养, 取菌液提取质粒DNA, 酶切鉴定重组体, 并送北京三博远志生物工程公司测序鉴定SV40T基因插入的方向及DNA序列, 插入方向正确的重组体记为pcDNA3.1/HKSV.

2 结果

2.1 pMT/HK质粒构建与鉴定

H⁺/K⁺ATPase β亚基启动子扩增产物用10 g/L琼脂糖凝胶电泳, 结果可见约1060 bp的DNA条带(图1), pMT/HK质粒用XbaⅠ、BamHⅠ双酶切电泳, 可见到1与2.6 kb的两条DNA条带, 与预期结果一致(图2).

图1 PCR电泳结果. 1: PCR产物; 2: PCR产物纯化; M: DL 15 000 DNA Marker.

图2 pMT/HK酶切鉴定. 1: T载体; 2: PCR产物; 3-4: pMT/HK质粒酶切后产物; M: DL15 000 DNA Marker.

2.2 pcDNA3.1/HKSV质粒构建与鉴定

pcDNA3.1/HKSV用XbaⅠ、BamHⅠ双酶切电泳, 可见到约1, 2.7与5.4 kb的3条DNA条带; 用XbaⅠ、KpnⅠ双酶切电泳, 可见到约3.7与5.4 kb的两条DNA条带; 用BamHⅠ单酶切电泳, 可见到2.7与6.4 kb的2条DNA条带; 用EcoRⅠ单酶切, 只见到约9.1 kb的1条DNA条带, 酶切电泳结果均与设计一致(图3).

图3 pcDNA3. 1/HKSV酶切鉴定. 1: XbaⅠ、BamHⅠ双酶切; 2: XbaⅠ、KpnⅠ双酶切; 3: BamHⅠ单酶切; 4: EcoRⅠ单酶切; M: DL15 000 DNA Marker.

2.3 DNA测序结果

pMT/HK测序显示, PCR扩增的H⁺/K⁺ATPase β亚基启动子长度为1057 bp(-1057～-1), 测序结果与Canfield et al[1]发表序列经NCBI网站的Blast 2 sequences 对比分析, 发现在文献起始密码子上游764与765 bp之间有AC碱基的插入; -501与-634 bp之间存在碱基重复序列, 并且不同小鼠扩增产物测序以及与文献序列之间比对发现存在1-2个重复序列的不同, 推测此段序列可能为多态性位点, H⁺/K⁺ATPase β亚基启动子测序结果如下:TCTAGAGCTCTTTCCTCTGGGTCTGTTTGGTGGAACACACAGTTCTGTGTTCTCTCTAGAGCTCTTTCCTCTGGGTCTGTTTGGTGGAACACACAGTTCTG TGTTCTCACACAGTAGAGAAACAGCCTCAGCCCTCCTCCCACAGACACACGATCAGGTCATGCTGACAATCCATGTATGGTCCAGACCCTACTCATGCTTCCAG AACCTCAGAGGTGGAGTTAGGGAAATGGAGCTTTAAAAAGCTCAGTAATAACTCAGCATTTTGTTCACACATGGAGCGCCTCCATGCTCACTGAGAGACAATGG CTGTGACTCTAAGATACCTTCATGCTCTTGGTTGCCAGACCCCAGGCTTGGGTTTGCATACACAAGCTAGCTCTAACCACCCAGGTAATTTCAGGTGCCCAGAC CTCTGACTCCAGGCCTACAGGCAATTTCCAGGCCCTGAATACCTAGATCCAGGTTTGTCTAACATGCTCTCTCTCTCTCTCTCTCTCTAAATATATATATATAT ATATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATATATATATATATATATATATATATATATATATATATATATACACATGGGTAT GCATATCTCCACAGGAGAATACTGAGGACAGGGCTGTGGGGCCACTCCAGGTAGTTGTAGGCACACTTTAAGCACCTTCTCCACCATCTTGAGGAGGAGGGGAG TCTCCAGGAAGCAGTTCGAGGTCCCAGGGACCTGAGTGGTGAGGTGGCAGGTCTCAGGCCTAGGGCATTTGACCTAGAAGCTGCCAGTATGTGTTTTCCCTCCT GAAGGGCAGGTGAGGCACAACCCAGAGGCTGTTCACATCAGACATGCTTCCCTTCAGCTAACATCAGGGTAGATGAAGTTGCCAGCCAAGGGCACCCCAAGGAC CAACTGACTTCTGGGACAGCGGAGGGCAGATAGCAAGCAAGCTCCAACCCTCCCTTGTGTTTGTAGAGGCGATAGTAGAGAACTGATAGCCGGTTCTGATGCCT TTGGCCTCACACAGAGGAGACTATAAGCCCTAGAGGACGCTTCCTGGGCCCAGTCCAGGCAAGCAGGAGAGGAC

注: 下划线为引物序列, 黑色区示AC碱基插入部位, 灰色区示重复碱基序列. pcDNA3.1/HKSV质粒中SV40T基因测序结果与NCBI公布的NC_001669序列完全一致.

3 讨论

SV40病毒属乳多空病毒科(papovaviridae)多瘤病毒属(polyomavirus), 其基因组包括5224 bp, 为双股环状DNA, 在病毒DNA复制前编码2种转化蛋白, 即大T抗原(Tag)和小T抗原(tag). 野生型SV40T基因全长2473 bp, 2个外显子连接编码序列长为2127 bp. Tag是一种磷酸化蛋白, 具有ATP酶和DNA解旋酶活性, 能使蛋白质丝氨酸、苏氨酸残基磷酸化、ADP核糖基化和乙酰基化, 另外还有活化宿主细胞核糖体基因、诱导DNA合成、修饰蛋白质合成起始因子等作用, 在细胞转化中起决定性作用, 为细胞转化所必需[2-9]. tag是非磷酸化蛋白, 对细胞转化并非必需, 但可起加强转化作用[10]. SV40T基因除用于细胞转化之外, 还发现与细胞增殖及多种肿瘤的产生相关, 并被广泛应用于转基因动物肿瘤模型的建立研究[11-20]. 如Kim et al[21]构建的共表达SV40T/抗利尿激素2转基因小鼠产生了脑瘤与淋巴瘤; Sun et al[22]将SV40T基因导入小鼠受精卵建立了前列腺癌的转基因鼠系; Nabarra et al[23]建立的在L丙酮酸激酶启动子调控下的SV40T转基因小鼠首先发生胸腺增生, 进而出现恶性胸腺瘤; Thompson et al[24]建立的CEA promotor/SV40T转基因小鼠后代在幽门处100%产生肿瘤, 37 d在小鼠胃黏膜层可观察到细胞萎缩, 50 d肿瘤可至黏膜下层, 100-130 d小鼠皆因幽门阻塞而死亡, 但由于CEA promotor/SV40T转基因小鼠基因表达的随机性, 在5000多只子鼠中仅发现一只胃癌转基因小鼠, 因此建立在胃组织特异细胞中表达的SV40T基因真核表达载体, 将为转基因小鼠及胃癌动物模型的建立提供稳定、可靠的分子工具.

胃癌是消化道恶性肿瘤中最多见的癌种, 死亡率居恶性肿瘤之首位, 建立胃癌肿瘤动物模型可为胃癌的早期诊断、治疗和发病机制的研究提供非常有用的实验动物模型[25-28]. Gordon et al研究了H⁺/K⁺ATPase β亚基在胃组织中的作用, 并建立了在胃壁细胞中特异性表达hGH, INF, SV40T的转基因动物模型[29-30], 证实了在H⁺/K⁺ATPase β亚基启动子作用下, hGH, INF, SV40T仅在小鼠胃壁细胞中表达, 在小肠、肝、幽门、贲门、大肠等处均不表达, 为胃组织特异性表达载体的构建提供了直接的理论依据. 我们成功构建了在小鼠胃壁细胞中特异性表达SV40T基因的真核表达载体, 为进一步转基因肿瘤动物的建立与研究奠定了基础.

评论

背景资料

转基因肿瘤动模型可为肿瘤发生机制的研究提供十分有用的实验动, 近年来其研究越来越受到关注. SV40(simianvirus40)属乳多空病毒科多型瘤病毒属中的一种小DNA肿瘤病毒, 具有转化动物细胞和诱发肿瘤的特性. SV40T转基因小鼠模型的发瘤效率很高, 利用SV40T基因已建立了小鼠乳腺癌、脑脉络丛乳头状瘤、胰腺癌等转基因动物模型. 建立在特定细胞中表达SV40T的转基因小鼠将可能诱发特定肿瘤的产生, 提供相应的肿瘤动物模型.

创新盘点

H⁺/K⁺ATP酶基因在胃壁细胞中特异性表达, 我们构建在H⁺/K⁺ATPaseβ基因启动子调控下的SV40T特异性表达载体将为转基因小鼠及胃癌动物模型的建立提供稳定、可靠的分子工具.

同行评价

本文为建立转基因小鼠及胃癌动物模型而构建了定向表达载体, 为进一步研究致癌机制建立了实验基础, 意义较大.

备注

编辑:王晓瑜 电编:张敏

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