血清类粘蛋白2下调乙型肝炎病毒表面抗原基因启动子I转录活性的研究

摘要

目的: 探讨血清类粘蛋白2(ORM2)对乙型肝炎病毒(HBV)表面抗原基因启动子Ⅰ(HBV-SP Ⅰ)转录的激活作用.

方法: 以我实验室前期得到的HBV-SP Ⅰ的酵母单杂交系统筛选结果为基础, 利用生物信息学技术确定ORM2的基因编码区域, 聚合酶链反应(PCR)扩增ORM2编码基因, 克隆至真核表达载体pcDNA3.1(-)中, 构建pcDNA3.1(-)-ORM2载体; 将该质粒与SP Ⅰ的氯霉素乙酰转移酶(CAT)报告载体pCAT3-SP Ⅰ共转染肝癌细胞系HepG2细胞系, 并以pCAT3-SP Ⅰ单独转染HepG2细胞系作为对照, 酶联免疫黏附法(ELISA)检测CAT的表达活性.

结果: pCAT3-SP Ⅰ在HepG2细胞中能够启动CAT的表达; 共转染实验中pCAT3-SP Ⅰ+pcDNA3.1(-)-ORM2组CAT的表达活性较pCAT3-SP Ⅰ下降了81.9%.

结论: ORM2蛋白具有对HBV-SP Ⅰ的反式抑制作用. 本实验验证了我室利用酵母单杂交技术筛选HBV-SP Ⅰ特异结合蛋白的结果, 为进一步了解HBV-SP Ⅰ的转录调控机制及其与SP I结合的反式作用因子提供了新的线索.

关键词: N/A

Down-regulating effect of orosomucoid 2 on preS1 promoter of hepatitis B virus

Yuan Hong, Jun Cheng, Qian Yang, Yan Liu, Jian-Jun Wang

Yuan Hong, Jun Cheng, Qian Yang, Yan Liu, Jian-Jun Wang, Gene Therapy Research Center, Institute of Infectious Diseases, the 302 Hospital of PLA, Beijing 100039, China

Supported by: Grants from the National Natural Scientific Foundation, No. C03011402, No. C30070689; and the 9.5 Research and Technique Foundation of PLA, No. 98D063; and the Launching Foundation for Student Studying Abroad of PLA, No. 98H038; and the 10.5 Youth Research and Technique Foundation of PLA, No. 01Q138; and the 10.5 Research and Technique Foundation of PLA, No. 01MB135.

Correspondence to: Dr. Jun Cheng, Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuanzhong Road, Beijing 100039, China. cj@genetherapy.com.cn

Received: November 13, 2003
Revised: December 1, 2003
Accepted: December 16, 2003
Published online: April 15, 2004

Abstract

AIM: To investigate activity of orosomucoid 2 (ORM2) on preS1 promoter (SP Ⅰ) of hepatitis B virus (HBV).

METHODS: Yeast one-hybrid system was employed in screening of DNA-binding proteins specifically recognizing HBV-SP I sequence, in which ORM2 was identified in GenBank by bioinformatics. For further studying the interaction between ORM2 and HBV-SP Ⅰ, the sequence of ORM2 was amplified from HepG2 genome by polymerase chain reaction (PCR) technique, which was then cloned into pcDNA3.1(-) expression vector. The HepG2 cell line was transfected by pCAT3- SP Ⅰ, and co-transfected by pCAT3-SP Ⅰ and pcDNA3.1(-)-ORM2, respectively. The chloramphenicol acetyltransferase (CAT) activity was detected by an enzyme-linked immunosorbent assay (ELISA) kit.

RESULTS: pCAT3-SP Ⅰ had higher activity of CAT than pCAT3-basic by ELISA kit. The expression of CAT from pCAT3-SP Ⅰ was increased 81.9%, as compared with that in the co-transfection of pCAT3-SP Ⅰ and pcDNA3.1(-)-ORM2.

CONCLUSION: Cell transfection and ELISA technology are successfully used to prove the results from yeast one-hybrid system, which brings some new clues for studying the specific binding proteins of HBV- SP Ⅰ and its transcriptional regulation mechanism.

Key Words: N/A

0 引言

乙型肝炎病毒(HBV)是一种严重危害人类健康的致病因子. 他感染人体除引起急慢性乙型肝炎和肝硬化外, 还与肝癌的发生有密切关系[1-4]. HBV属嗜肝DNA病毒, 目前在其基因组中已发现并鉴定的顺式元件有4个启动子、2个增强子和糖皮质激素应答元件. 其中表面抗原基因含有两个串联的启动子SP I和SP Ⅱ. SP I (2 219-2 780 nt)的主要作用是调节2.4 kb mRNA的转录, 编码表面抗原大蛋白[5-8]. 有研究[9]证实, SP I含有典型TATA盒序列, 是HBV转录调节的重要组分, 也是HBV嗜肝性的重要原因之一. 为了寻找与SP I结合的肝特异性转录作用因子, 我室[10]应用酵母单杂交体系[11-23], 以SP I核心序列为"诱饵", 对人肝细胞cDNA文库成功进行了筛选, 得到了12个结合蛋白基因. 本实验为了对SP Ⅰ酵母单杂交的筛选结果进行验证, 选取了其中的一个反式作用因子基因-血清类粘蛋白2(ORM2)为研究对象, 利用生物信息学手段对ORM2基因编码序列进行分析、克隆, 与真核表达载体pcDNA3.1(-)连接, 构建了表达质粒; 与SP Ⅰ的CAT报告载体pCAT3-SPⅠ共转染, 旨在研究ORM2对SP Ⅰ是否具有激活作用, 使下游CAT基因的表达发生变化, 这一结果也为研究HBV的转录调节机制提供新的理论依据.

1 材料和方法

1.1 材料

HepG2细胞及感受态E.coli JM109(本室保存), pcDNA3.1(-)真核表达载体(Invitrogen); Lipofectamine PLUS 转染试剂(Gibco), mRNA Purification试剂盒(Amersham Pharmacia Biotech), PCR-Select cDNA Subtraction试剂盒, 50×PCR Enzyme Mix、Advantage PCR Cloning试剂盒(Clontech), High Pure PCR Product Purification试剂盒(Boehringer Mannheim), T7、SP6通用引物pGEM-Teasy载体(Promega). 真核表达载体及pCAT3-SP I重组质粒为本室构建.

1.2 方法

1.2.1 基因编码序列分析: 根据GenBank中ORM2的基因组序列, 确定ORM2的翻译起始子及终止密码子. 设计并合成引物, 在上下游引物的5'-端分别加上EcoR I和Xho I酶切位点序列. 上游引物: 5'-GAA TTC ATG GCG CTG TCC TGG GTT CT-3'; 下游引物: 5'-CTC GAG GAT CCA AGG CTG TGT CCT GC-3', 全长共625 bp.

1.2.2 质粒构建: 以HepG2细胞基因组为模板, 进行聚合酶链反应(PCR)反应, PCR产物经1%琼脂糖凝胶电泳, 切胶, 玻璃奶回收DNA片段, 纯化. 在T4 DNA连接酶的作用下, 与pGEM-T载体连接, 转化大肠杆菌DH5, 挑取在选择平皿(Amp, X-gal/IPTG)生长的白色菌落提取质粒, 经酶切(EcoR I/Xho I)及测序鉴定. EcoR I/Xho I双酶切重组质粒pGEM-T-ORM2, 玻璃奶纯化回收酶切产物, 定向克隆至pcDNA3.1(-)载体, 构建成重组质粒pcDNA3.1(-)-ORM2. 经双酶切及菌落PCR鉴定连接产物, 试剂盒法提取质粒.

1.2.3 细胞培养及pCAT3-SPⅠ转染HepG2细胞系: HepG2细胞系在含10%胎牛血清的DMEM培养液中生长. 细胞生长至50%-80%时采用脂质体转染法, 具体转染方法参照FuGENE6说明书进行, 质粒pCAT3-SPⅠ转染48 h后收获细胞.

1.2.4 共转染实验: 细胞生长至50%-80%融合度时, 质粒pCAT3-SPⅠ与pcDNA3.1(-)-ORM2共转染HepG2细胞系. 具体转染方法参照FuGENE6说明书进行, 转染48 h后收获细胞.

1.2.5 CAT表达量的检测: 将收获的细胞进行裂解, 提取上清液, 取200 L用于检测CAT的表达量. 具体方法严格按照CAT ELISA试剂盒说明书进行, 在415 nm光波下测吸光度A值.

2 结果

2.1 pcDNA3.1(-)-ORM2重组质粒的构建

利用自行设计的引物成功扩增出ORM2的基因编码序列, 经1%琼脂糖凝胶电泳分析显示扩增片段约625 bp, 与预期片段符合(图1). 将扩增产物与pGEM-T载体连接, 双酶切鉴定, 1%琼脂糖凝胶电泳分析显示两条带(3 000 bp的pGEM-T载体和625 bp的ORM2 DNA片段), 与预期一致(图2). 将双酶切产物与pcDNA3.1(-)载体连接, 双酶切鉴定如图3所示(5 400 bp的pcDNA3.1(-)载体和625 bp的ORM2 DNA片段), DNA测序结果和GenBank中ORM2的基因序列完全一致.

图1 以HepG2基因组DNA为模板, PCR扩增ORM2序列.

图2 pGEM-T-ORM2双酶切鉴定(EcoR I/Xho I).

图3 pcDNA3. 1(-)-ORM2双酶切鉴定(EcoR I/Xho I).

2.2 pCAT3-SPⅠ重组质粒的瞬时转染及报告基因CAT表达的检测

将重组质粒pCAT3-SPⅠ转染HepG2细胞, CAT ELISA结果如表1所示, 具有启动子活性.

表1 pCAT3-SP Ⅰ转染HepG2细胞后CAT的表达(A值,mean±SD).

组别	质粒	吸光度
阴性对照组	pCAT3-basic	0.450±0.012
阳性对照组	pCAT3-promoter	0.875±0.022
实验组	pCAT3-SP Ⅰ	2.136±0.051

2.3 pCAT3-SPⅠ与pcDNA3.1(1)-ORM2重组质粒共转染及CAT表达的检测

将pCAT3-SP Ⅰ与pcDNA3.1(-)-ORM2共转染HepG2细胞时, CAT的表达较pCAT3-SPⅠ单独转染明显降低, 如表2所示, 说明ORM2对HBV-SPⅠ具有反式抑制作用.

表2 pCAT3-SP Ⅰ与pcDNA3.1(-)-ORM2共转染HepG2后CAT的表达(A值, mean±SD).

组别	质粒	吸光度
阴性对照组	pCAT3-basic	0.338±0.002
阳性对照组	pCAT3-promoter	0.612±0.007
实验对照组	pCAT3-SP Ⅰ	2.143±0.028
共转染组	pCAT3-SP Ⅰ+pcDNA3.1(-)-ORM2	0.389±0.075

3 讨论

SP I是HBV的一个重要转录元件, 调节HBV表面抗原大蛋白的表达. 对携带HBV DNA的转基因小鼠及感染HBV的黑猩猩模型的研究[24-25]表明, 表面抗原主蛋白可在不同的组织中表达, 而表面抗原大蛋白只在肝内特异发现, 这一结果强烈提示: 表面抗原大蛋白的表达具有肝特异性, SP I在肝特异性表达中起着关键作用. Raney et al[6]通过应用瞬时转染分析、体外RNase保护分析等方法, 对这一现象的原因进行了研究, 结果显示, 肝细胞核因子1(HNF1)与SP I的结合可能是主要原因. 将该因子去除后, SP I的转录活性下降了20倍. 同时可能还有其他肝特异因子参与调节. 为了寻找新的肝特异结合蛋白, 我室以SP I核心序列为"诱饵", 整合入酵母基因组, 构建了酵母报告株; 经加入人肝cDNA文库进行筛选, 得到了12个双阳性克隆, 共表达10种蛋白, 其中有血清类粘蛋白2、丝氨酸脱水酶、人肝细胞生长因子样蛋白等已知功能蛋白及3个未知功能蛋白. 为了对这一结果进行验证并深入研究HBV的转录调控机制, 我们选取了其中的一个已知功能基因-ORM2为研究对象, 以CAT为报告基因, 在真核细胞内对二者的相互作用进行了共转染验证.

人血清类粘蛋白又称为1酸糖蛋白(1-acid glycoprotein, AGP), 是分子量为41-43 kDa, 等电点(pI)为2.8-3.8的糖蛋白[26]. 在人、鼠和其他物种中, 他都是一种主要的血浆急性期反应蛋白. 当有组织损伤或感染时, 在糖皮质激素和细胞因子的联合作用下, 其表达水平明显升高, 可作为感染性肠炎的诊断标志之一[27]. 他的氨基酸序列首先从人的血浆中分离得到, 从不同个体的血浆中分离得到的ORM序列具有很大的异质性[28]. 其中ORM2编码基因位于ORM1下游约3.3 kb处, 由2个分离的基因座组成; Northern blot杂交检测结果显示[29], 人肝脏中的ORM mRNA主要来自ORM1编码基因的转录, ORM2在肝脏中的表达水平很低. 尽管对ORM的结构研究早已开始, 但目前对他的生物学功能了解的仍不十分清楚. 一般认为, 他在血浆中可以结合和携带亲脂性药物, 使药物发挥效用; 依赖碳端氨基酸, 发挥免疫应答介导和结合活性; 对ORM转基因鼠的研究可以在小鼠体内了解与ORM结合药物的效果, 揭示免疫反应中应答元件和组织特异性元件的功能[30].

本实验的主要目的是验证前期酵母单杂交的筛选结果, 在共转染实验中pCAT3-SPⅠ和pcDNA3.1(-)-ORM2组的CAT的表达活性较pCAT3-SPⅠ单独转染下降了81.9%, 说明ORM2可与HBV-SP I结合, 且对SP I的转录活性有明显的抑制作用. 尽管这一结论与体内的情况不符, 因为在体内ORM2主要分布于血浆中, 与HBV-SP I的核内分布存在着一定的区室阻隔. 但这一结果有可能为今后HBV的治疗开辟一条新途径.

备注

编辑: N/A

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