肝癌 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-11-15; 11(11): 1674-1676
在线出版日期: 2003-11-15. doi: 10.11569/wcjd.v11.i11.1674
小鼠甲胎蛋白基因的克隆真核表达载体构建及表达鉴定
田耕, 易继林
田耕, 易继林, 华中科技大学同济医学院附属同济医院普外科 湖北省武汉市 430030
田耕, 男, 1971-03-18生, 陕西省宝鸡市人, 汉族, 博士, 主治医师.
通讯作者: 田耕, 430030, 湖北省武汉市汉口解放大道1095号, 华中科技大学同济医学院附属同济医院普外科. geng_tian707@hotmail.com
电话: 027-83663402
收稿日期: 2003-03-08
修回日期: 2003-03-20
接受日期: 2003-03-25
在线出版日期: 2003-11-15

目的

克隆小鼠甲胎蛋白(AFP)基因, 构建小鼠AFP真核表达载体并进行表达鉴定.

方法

从Hepa1-6细胞中提取总RNA进行RT-PCR, 克隆出小鼠AFP基因, 亚克隆于pcDNA3.1载体, 重组阳性克隆进行酶切和测序鉴定. 重组质粒瞬时转染CHO-K1细胞, Western blot检测小鼠AFP的表达.

结果

利用RT-PCR从Hepa1-6细胞总RNA中成功克隆出1.8 kb的小鼠AFP基因, 重组阳性克隆经酶切和测序鉴定证实目的基因已正确插入pcDNA3.1载体中, Western blot结果证实重组质粒pmAFP能够在CHO-K1细胞中正确表达.

结论

小鼠AFP真核表达载体构建成功, 为进一步研究其在肝癌免疫治疗中的作用奠定了基础.

关键词: N/A
引文著录: 田耕, 易继林. 小鼠甲胎蛋白基因的克隆真核表达载体构建及表达鉴定. 世界华人消化杂志 2003; 11(11): 1674-1676
Gene cloning of murine α-fetoprotein gene and construction of its eukaryotic expression vector and expression in CHO cells
Geng Tian, Ji-Lin Yi
Geng Tian, Ji-Lin Yi, Department of General Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Corresponding author: Geng Tian, Department of General Surgery , Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. geng_tian707@hotmail.com
Received: March 8, 2003
Revised: March 20, 2003
Accepted: March 25, 2003
Published online: November 15, 2003

AIM

To clone murine AFP gene, to construct the eukaryotic expression vector of AFP and express it in CHO cells.

METHODS

Total RNA was extracted from Hepa1-6 cells, then the murine AFP gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant vector was transformed into E.coli. DH5α, the positive clones were selected and plasmid DNA was identified by restriction enzyme analysis and sequencing. After transient transfection of CHO-K1 cells with the recombinant vector, Western blotting was used to detect the expression of AFP.

RESULTS

The 1.8 kb murine α-fetoprotein gene was successfully cloned from the total RNA of Hepa1-6 cells. Result from restrictiive enzyme analysis and sequencing showed that the murine α-fetoprotein gene was successfully inserted into pcDNA3.1. Result from Western blotting showed that the recombinant vector could express murine α-fetoprotein in CHO-K1 cells.

CONCLUSION

The successfully constructed eukaryotic expression vector of murine α-fetoprotein could provide a basis for the research of immunotherapy for hepatocellular carcinoma with pmAFP.

Key Words: N/A
0 引言

肿瘤生物治疗[1]是继手术[2-13]、放疗[2,14,15]、化疗 [2,16-18]后的一项有效的辅助治疗手段, 其中, 肿瘤疫苗又为肿瘤生物治疗的热点[19,20]. 目前, 肿瘤疫苗主要包括全细胞疫苗、多肽疫苗、基因工程疫苗、DNA疫苗[21]、抗独特型抗体疫苗[22]等多种形式. 其中, DNA疫苗技术是1990年代初发展起来的一项新兴免疫技术, 因其独有的特点而更受瞩目. 肝细胞肝癌(HCC)作为常见消化系统肿瘤, 复发率高, 预后较差[23-33]. AFP作为肝癌的相关抗原存在CTL免疫显性表位, 完全可以作为肝癌免疫治疗的靶的[34,35]. 我们构建了编码小鼠AFP的真核表达载体, 为进一步研究基于AFP的肝癌免疫治疗奠定了基础.

1 材料和方法
1.1 材料

小鼠肝癌细胞系Hepa 1-6由第二军医大学王皓博士惠赠; CHO-K1细胞由中国典型培养物保藏中心提供; E.coli. DH5α菌种由华中科技大学同济医学院蔡俐琼硕士惠赠; 真核表达载体pcDNA3.1/myc-His购自Invitrogen公司; RT-PCR试剂盒(Ver.2.1)、Ex Taq高保真Taq酶、限制性内切酶、T4DNA连接酶和DNA Marker (DL2000)购自宝生物工程(大连)有限公司; Trizol试剂和RPMI1640培养基为Gibco公司产品、胎牛血清为Hyclone公司产品; 脂质体Lipofectamine 2000和Opti-MEM 培养基为Invitrogen公司产品; 质粒小量制备试剂盒和琼脂糖凝胶核酸纯化回收试剂盒为Omega公司产品; 羊抗人AFP多克隆抗体购自Sant Cruz 公司; HRP标记的抗羊二抗为北京中山生物工程公司进口分装产品; ECL显色剂为Pharmacia公司产品; 其余试剂均为国产或进口分析纯试剂.

1.2 方法

1.2.1 小鼠AFP基因的克隆 培养Hepa 1-6细胞至对数期, 收集细胞, 细胞数应在(5-10)×106, 用Trizol试剂提取细胞总RNA. 为了克隆包括分泌信号在内的AFP全长基因, 设计的引物为P1、P2, P1: 5'-CTC AGGAATTCGCCATGAAGTGGATCACA-3', 在5'端引入酶切位点Eco RI; P 2: 5'-CTCTGCTCTAGATTACTCGAGAACGCCCAAAGCATCACG-3', 在3'端引入酶切位点Xba I. 引物由上海博亚生物工程公司合成. 利用RT-PCR试剂盒反转录出cDNA第1链, 引物采用Oligo dT, 条件为: 42 °C 30 min、99 °C 5 min、5 °C 5 min. 采用Ex Taq高保真Taq酶进行随后的PCR. PCR条件为: 94 °C预变性2 min、94 °C 3 0 s、60 °C 30 s、72 °C 2 min, 30个循环, 末次72 °C延伸5 min. 取PCR产物3 μl进行琼脂糖凝胶电泳, 观察结果.

1.2.2 重组质粒的构建、酶切及测序鉴定 克隆的小鼠AFP基因经Eco RI和Xba I同时双酶切后, 琼脂糖凝胶电泳回收、纯化; 质粒pcDNA3.1/myc-His同样经Eco RI和Xba I同时双酶切后, 回收纯化. 目的基因与质粒按3: 1的比例混合, 加入T4DNA连接酶进行连接反应(18 °C, 16 h). 构建的质粒命名为pmAFP. 将上述连接产物转化大肠杆菌DH5α, 涂平板, 氨苄青霉素筛选阳性菌落. 挑取单个菌落培养, 小量质粒制备. pmAFP用Eco RI和Xba I进行单酶切或双酶切鉴定. 酶切鉴定正确的质粒, 挑取相应菌落培养后送上海博亚生物工程公司进行双向测序.

1.2.3 pmAFP转染CHO细胞与蛋白表达检测 利用脂质体Lipofectamine 2000 瞬时转染CHO-K1细胞, 按说明书进行操作, 同时做空载体对照. 在6孔板每孔中加入5×105 个CHO-K1细胞, 24 h 后加入分别稀释于Opti-MEM 培养基的4 μg质粒和10 μl Lipofectamine 2000, 48 h后裂解细胞, 提取蛋白. 蛋白质经电泳后, 半干法转印至硝酸纤维素膜上, 一抗为羊抗人AFP多克隆抗体, 然后用HRP标记的抗羊二抗孵育, ECL显色.

2 结果
2.1小鼠AFP基因的克隆

提取的总RNA A260/A280比值为1.885, 表明总RNA较纯. 以反转录的cDNA为模板, 用设计引物进行PCR扩增, 所得特异性条带均与预期长度为1.8 kb的目的基因相符(图1).

图1
图1 RT-PCR获得小鼠AFP基因. M: DNA marker; 1: Murine AFP gene.
2.2 重组质粒酶切及测序鉴定

证明重组质粒带有相应的目的基因(图2). 对重组子进行测序, 结果与Gene bank Blast 比对, 本实验克隆出的小鼠AFP基因与Gene bank所给出的小鼠AFP基因有99.7%的碱基相同, 共有5个碱基不同, 其中有两个碱基为密码子的第3位碱基, 未改变所编码的氨基酸.

图2
图2 重组质粒pmAFP酶切鉴定. M: DNA marker(DL2000); 1: pmAFP (Eco RI + Xba I); 2: pcDNA3.1(Eco RI); 3: pmAFP(Eco RI).
2.3 Western blot检测蛋白表达

利用脂质体Lipofectamine 2000 (Invitrogen) 瞬时转染CHO-K1细胞, 48 h后提取蛋白. Western blot检测可检出转染细胞中有 M r 70 000的特异性蛋白条带(图3), 表明小鼠AFP基因在真核细胞内得到表达.

图3
图3 Western blot检测结果. 1: CHO-K1/pmAFP; 2: Hepa 1-6; 3: CHO-K1/ pcDNA3.1.
3 讨论

DNA疫苗作为一种新的免疫接种手段, 问世不久就在感染性疾病及肿瘤的防治中显示出巨大的应用潜力. 然而, 以往的研究显示, 肝癌抗原性弱, 缺少特异性抗原, 这成为肝癌免疫治疗的一个主要难点. 随着基础免疫学与肿瘤免疫学的迅猛发展, 人们已认识到: (1)细胞免疫尤其是CD8+的Ｔ细胞介导的特异性MHC-I类分子限制性细胞免疫功能在抗肿瘤免疫中起决定作用; (2)特异性CD8+Ｔ细胞激活及杀伤靶细胞的前提是TCR同时识别MHC-I类分子与8-10个氨基酸残基组成的CTL表位肽. 在此理论基础上, 寻找肿瘤特异性或相关性抗原及其CTL表位用以构建CTL疫苗的研究方兴未艾并取得重大突破, 对黑色素瘤、结肠癌、乳腺癌的免疫治疗研究已进入I, II期临床. 运用这种方法对人AFP的研究发现, 人的AFP存在4个CTL表位, 这些免疫显性表位能诱导T细胞产生AFP特异性的CTL, 产生针对AFP的抗肝癌免疫力[34,35]. 研究已证实, 基于AFP的免疫治疗在小鼠体内能够诱导T细胞产生AFP特异性的CTL, 产生针对AFP的抗肝癌免疫力.

我们克隆出的小鼠AFP基因共有1818个碱基, 与Gene bank所给出的小鼠AFP基因有99.7%的碱基相同, 共有5个碱基不同, 其中有两个碱基为密码子的第3位碱基, 未改变所编码的氨基酸. 存在差异的原因可能是Gene bank所给出的小鼠AFP基因来源于小鼠13 d胚胎的肝脏.

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