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100023 北京市2345信箱	世界华人消化杂志 2002年2月15日;10(2):141-144
Email: wcjd@public.bta.net.cn	世界华人消化杂志 ISSN 1009-3079 CN 14-1260/R
http:// www.wjgnet.com	版权归世界胃肠病学杂志社

⊙病毒性肝炎⊙

截短型乙型肝炎病毒表面抗原中蛋白上调c-myc基因表达的研究

韩萍，刘妍，成军，王刚，陆荫英，李克，李莉

韩萍，刘妍，成军，王刚，陆荫英，李克，李莉，中国人民解放军第302医院传染病研究所基因治疗研究中心北京市 100039
韩萍，女，1966-06-01生，陕西省蓝田县人，汉族. 1990年新疆医科大学毕业，学士学位，从事病毒性肝炎的临床治疗及实验研究.
军队回国留学人员启动基金资助课题，No.98H038和国家自然科学基金资助课题，No.C30070689
项目负责人 成军，100039，北京丰台路26号，中国人民解放军第302医院传染病研究所基因治疗研究中心. cj@GeneTherapy.com.cn
电话: 010-66933392 传真: 010-63801283
收稿日期 2002-01-15 接受日期 2002-02-05

The expression of oncogene c-myc upregulated by c-terminal ly truncated middle surface protein of hepatitis B virus

Ping Han, Yan Liu, Jun Cheng, Gang Wang, Yin-Ying Lu, Ke Li, Li Li

Ping Han, Yan Liu, Jun Cheng, Gang Wang, Yin-Ying Lu, Ke Li, Li Li ，Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospit al of PLA, Beijing, China
Supported by grants of Returned Scholarship of General Logistics Departm ent of PLA,No.98H038, and National Natural Science Foundation of China,No.C30070 689
Correspondence to: Jun Cheng, Gene Therapy Research Center, Inst itute of Infectious Diseases, The 302 Hospital of PLA, 26 Fengtai Road, Beijing, 100039, China. cj@GeneTherapy.com.cn
Received 2002-01-15 Accepted 2002-02-05

Abstract
AIM:To construct the cDNA library of genes transactiva ted by c-terminally truncated middle surface protein of hepatitis B virus(MHBs ^t) using suppression subtractive hybridization (SSH) technique and to further investigate the expression of oncogene c-myc upregulated by MHBs^t.

METHODS:We constructed a recombinant expression plasmid containi ng c-terminally truncated middle surface protein of hepatitis B virus. The reco mbinant plasmid pcDNA3.1(-)-Mt and the empty vector pcDNA3.1(-) were transien tly transfected into HepG2 cells, respectively. The mRNA was extracted from HepG 2 cells transfected then the cDNA was synthesized. The tester cDNA was hybridize d with driver cDNA and underwent nested PCR twice with suppression subtractive h ybridization technique. The subtractive library was amplified with E.coli st rain. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR . The bioinformatic analysis showed some sequences may be involved in tumour dev elopment such as oncogene c-myc. The upregulated expression of oncogene c- myc was verified by immunoblotting.

RESULTS:The western blotting result indicated that the expressio n of c-myc gene was upregulated by MHBs^t.

CONCLUSION:The cDNA subtractive library of genes transactivated by MHBs^t was successfully constructed and MHBs^t could upregulate expression of oncogene c-myc,which provided a basis for clarifying the tumor developm ent mechanism of HBV.

Han P, Liu Y, Cheng J, Wang G, Lu Y, Li K， Li L. The expression of onc ogene c-myc upregulated by c-terminally truncated middle surface protein o f hepatitis B virus.Shijie Huaren Xiaohua Zazhi 2002;10(2):141-144

摘要
目的:应用抑制性消减杂交方法构建截短型乙型肝炎病毒表面抗原中蛋白（MHBs^t）反式调节基因的消减文库，用免疫印迹方法验证截短型乙型肝炎病毒表面抗原中蛋白对c-myc基因的上调表达.

方法:构建羧基末端截短的乙型肝炎病毒表面抗原中蛋白的真核表达载体pcDNA3.1(-)-Mt，分别以重组表达质粒pcDNA3.1(-)-Mt和空载体pcDNA3.1(-)瞬时转染HepG2细胞，提取细胞mRNA并逆转录为cDNA，用抑制性消减杂交技术，将实验组与对照组cDNA进行两次消减杂交及两次抑制性多聚酶链反应(PCR)，构建cDNA消减文库，并转染大肠杆菌进行文库扩增，随机挑选克隆PCR扩增后进行测序及同源性分析.生物信息学分析结果显示部分与肿瘤发生密切相关的基因，如癌基因c-myc.

结果:显示截短型乙肝病毒表面抗原中蛋白可以反式激活c-myc基因，并上调其表达.

结论:成功构建截短型乙肝病毒表面抗原中蛋白反式调节基因的消减文库，细胞原癌基因c-myc是MHBs^t反式激活作用的靶基因，为进一步阐明乙型肝炎病毒致癌的分子生物学机制提供理论基础.

韩萍，刘妍，成军，王刚，陆荫英，李克，李莉.截短型乙型肝炎病毒表面抗原中蛋白上调c-myc基因表达的研究.
世界华人消化杂志 2002;10(2):141-144

0 引言
乙型肝炎病毒（HBV）感染与肝细胞癌发生密切相关，病毒基因组编码的蛋白与宿主肝细胞之间的相互作用，可能是病毒致癌的重要分子机制.近年研究表明，乙型肝炎病毒表面抗原中蛋白（MHBs^t）能影响多种细胞信号转导途径，激活多种病毒及细胞基因启动子，具有广泛的反式激活作用^［^1-4^］.我们构建了羧基末端截短的MHBs^t的真核表达载体pc DNA3.1(-)-Mt，与空载体分别瞬时转染HepG2细胞，应用抑制性消减杂交技术筛选MHBs^t反式激活的靶基因，筛选的序列中含有与肿瘤发生密切相关的基因如c-myc，进一步用免疫印迹方法验证MHBs^t对c-myc基因表达的上调作用，结果显示c-myc 是MHBs^t反式激活作用的靶基因，为进一步阐明HBV致癌分子生物学机制提供依据.

1 材料和方法
1.1 材料大肠杆菌JM109、肝母细胞瘤细胞系HepG2细胞及含有双拷贝ayw亚型HBV全基因组的pCP10质粒为本室保存；克隆载体pCR2.1及真核表达载体pcDNA3.1(-)购于Invitrogen公司；Lipofectamine PLUS 转染试剂购于Gibco 公司；mRNA Purification试剂盒购于Amersham Pharmacia 公司；PCR-Select cDNA Subtr action Hybridization全套试剂盒购于Clontech公司；T7、SP6通用引物，工具酶及pGEM- Teasy载体购于Promega公司；c-myc单克隆抗体本室自制，由购自ATCC的1-9E10.2杂交瘤产生；辣根过氧化物酶标记羊抗鼠IgG购于北京中山公司.
1.2 方法 重组表达载体的构建及表达：以质粒pCP10中所含的HBV ayw亚型的DNA序列为模板，PCR扩增HBV表面抗原中蛋白截短型基因.以T-A克隆法，用T4 DNA连接酶将目的片段插入载体pCR2.1.将获得质粒pCR2.1-Mt和真核表达载体pcD NA3.1(-)分别用HindⅢ和ApaI双酶切，进行定向连接，产物转化DH5α感受态细胞，筛选抗氨苄青霉素阳性菌落，提取质粒，酶切鉴定含有MHBst基因的阳性克隆，命名为pcDNA3.1( -)-Mt. DNA测序由Takara公司完成.转染真核细胞并用ELISA方法检测基因的表达.
1.3 抑制性消减杂交 用Lipofectamine PLUS转染试剂将2μg pc DNA3.1(-)-Mt及pcDNA3.1(-)空载体分别转染35mm平皿HepG2细胞，48h后收获细胞并提取细胞mRNA.按抑制性消减杂交(suppression subtractive hybridization, SSH)常规方法，将转染了重组表达质粒及空载体的HepG2细胞cDNA分别标记为Tester和Driver，经Rs aI消化后，将Tester cDNA分为两份，分别连接不同的接头，然后与过量的Driver cDNA进行2次消减杂交和2次抑制性PCR扩增，将扩增产物与pGEM-Teasy载体连接，转化JM109感受态细胞，筛选阳性菌落.以T7/SP6引物进行菌落PCR扩增，证明含有插入片段（200-800bp ），测序.应用生物信息学将测得序列与GenBank数据库进行同源性分析.
1.4 细胞蛋白提取及免疫印迹 将pcDNA3.1(-)-Mt及pcDNA3.1( -)分别转染HepG2细胞，提取细胞裂解液50μl，分别取10μl进行12.5%十二烷基磺酸钠- 聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，电转移至硝酸纤维素膜.该膜经5%脱脂奶粉封闭后，分别与鼠抗人c-myc抗体和辣根过氧化物酶标记羊抗鼠IgG抗体各孵育1h，DA B显色，拍照.所有实验严格平行操作.

2 结果
2.1 重组质粒的酶切鉴定及表达用HindⅢ和ApaI双酶切，所得基因片段为620 bp（120+500bp）定向插入pcDNA3.1(-)载体后，分别用XhoI、XbaI、Eco RI和HindⅢ/ApaI酶切，释放出170bp、300bp、500bp和620bp条带见图1.DNA测序证实重组质粒含500bp核苷酸的目的基因，读码框架正确，保证其表达产物正确.将重组质粒pcDNA3.1 (-)-Mt瞬时转染真核细胞，表达出相应编码蛋白.应用报告基因β-半乳糖苷酶共转染实验显示，瞬时表达的病毒蛋白MHBs^t具有反式激活作用，能够激活SV40早期启动子/增强子，使其调控的下游lac Z基因表达增强^［⁴^］.

图1 pcDNA3.1(-)-Mt质粒酶切鉴定的电泳图谱
1：DL15000+DL2000；2-5：依次为XhoI、XbaI、EcoRI和HindⅢ/ApaI酶切pcDNA3.1(-)-Mt的图谱

2.2 SSH实验cDNA测序结果 在筛选到的94个阳性克隆中，随机挑选50个测序，与GenBank数据库进行同源性分析，结果显示其中4个克隆未检索到任何对应的相似序列，可能代表了某些新基因，进一步的分析和功能鉴定正在进行中. 另外46个克隆均与已知基因的部分序列高度同源（95%-100%），其中含有与细胞生长调节密切相关的序列如细胞原癌基因c-myc^［⁵^］.
2.3 癌基因c-myc表达结果 我们用免疫印迹方法观察MHBs^t是否反式激活癌基因c-myc的表达.Western blotting结果显示：转染了pc DNA3.1(-)空载体的HepG2细胞癌基因c-myc的表达与HepG2细胞本身c-myc的表达量相似，而转染了pcDNA3.1(-)-Mt的HepG2细胞癌基因c-myc的表达大大增强，提示M HBs^t对c-myc的表达有上调作用，c-myc基因是MHBs^t反式调节的靶基因（图2 ）.

图2 c-myc表达的Western Blotting图
1：转染了pcDNA3.1(-)-Mt的HepG2细胞；2：转染了pcDNA3.1(-)空载体的HepG2细胞；3：未转染质粒的HepG2细胞

3 讨论
HBV感染后，病毒与肝细胞之间相互作用的分子生物学机制还不十分清楚，其中病毒编码的蛋白能够影响肝细胞某些基因表达调控从而影响细胞生长调节可能是病毒致癌的主要因素^［^1-7^］.近年研究发现，从肝癌细胞系或肝癌组织，甚至是外周血中克隆出的截短型前S2/S基因表达产物羧基末端的截短型分子MHBst是一种具有广泛活性的转录激活因子 ^［^8-14^］，这种变异的病毒表面中蛋白，与野生型病毒比较，蛋白的羧基末端存在不同程度的缺失，由于结构的改变，MHBs^t具备内质网定位功能，而不象全长中蛋白那样进入高尔基复合体而分泌.正是MHBs^t在胞质中的滞留，才能够发挥其广泛的反式激活效应 .研究证实MHBs^t发生蛋白激酶C（PKC）依赖的磷酸化反应，前S2区域与PKCα/β结合，触发PKC依赖的c-Raf-1/MAP2-激酶信号传递链式反应，结果激活了转录因子同源/异源的病毒/细胞转录调节序列，参与病毒感染后的炎症反应和肝细胞癌的发生.因此研究MHBs^t的反式激活功能，有利于阐明HBV感染的慢性化和肝细胞癌形成的机制，同时也是探索 HBV感染治疗新途径一个重要的突破口.具有反式激活功能的截短位点是在一定的范围之内，本实验选择编码167位氨基酸残基以后的羧基末端截短分子-MHBs^t167，构建其真核表达载体，并证明在真核细胞中瞬时表达的MHBs^t蛋白具有反式激活作用^［^15-20^］ .抑制性消减杂交技术(SSH)是1990年代后期建立的一种基因克隆的新技术，可以快速有效地检测到差异表达的基因^［^21-25^］.我们用SSH方法成功地构建了HBV截短型中蛋白反式激活相关基因差异表达的cDNA消减文库，随机挑选50个克隆测序分析，结果显示其中4个克隆未检索到任何对应的相似序列，可能代表了某些新基因，对于这4个差异表达的未知序列，进一步的分析和功能鉴定正在进行中.另外46个克隆均与已知基因的部分序列高度同源（95%-100%），其中含有与细胞生长调节密切相关的序列如细胞原癌基因c-myc^［^26-32^］.获得的这些序列是否是真正的MHBs^t反式激活靶基因，我们进一步用免疫印迹方法得以证实，MHBs^t能上调c- myc基因的表达.C-myc基因是人的正常细胞基因组中一种高度保守的细胞癌基因^［^33-43^］，具有能够使正常细胞发生恶性转化的潜能.在大部分情况下，处于不表达状态或表达水平不足以引起细胞恶性转化.癌基因激活方式有多种，常见的如基因放大，基因突变，异源启动子表达，基因重排，基因过表达以及截短形式多肽的表达.本实验结果显示MHBs^t上调c-m yc基因的表达，对于阐明MHBs^t反式激活作用及其在HBV感染的慢性化及肝细胞癌发生之间的作用具有重要的理论意义.

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