乙型肝炎病毒X蛋白与去唾液酸糖蛋白受体2突变体相互作用的研究

摘要

目的

筛选并克隆鉴定人肝细胞中与乙型肝炎病毒(HBV)X蛋白(HBxAg)相互作用蛋白的基因, 明确HBxAg在HBV感染及致癌过程中的具体作用.

方法

用多聚酶链反应(PCR)法扩增HBxAg基因, 连接入酵母表达载体pGBKT7中构建诱饵质粒, 转化酵母细胞AH109并在其内表达, 然后与转化了人肝cDNA文库质粒的酵母细胞Y187进行配合, 在营养缺陷型培养基上进行双重筛选阳性菌落, PCR从中扩增出阳性目的片段并测序, 进行生物信息学分析. 根据GenBank中的序列信息设计引物, 从HepG2细胞的mRNA中逆转录出去唾液酸蛋白受体2(ASGPR2)突变体的完整序列, 克隆到另一酵母表达载体pGADT7中, 体外免疫共沉淀再次证明HBxAg与ASGPR2突变体的结合作用.

结果

成功克隆出HBxAg基因并在酵母细胞中表达, 配合后选出既能在四缺( SD/-Trp-Leu-His-Ade)培养基又能分解X-α-半乳糖(X-α-gal)变成蓝色的真阳性菌落41个, 其中有一个是ASGPR2的新突变体. HepG2细胞的mRNA中能逆转录出ASGPR2的全基因序列, 体外免疫共沉淀结果证实该突变体与HBxAg在体外也有结合作用.

结论

成功克隆出HBxAg的肝细胞结合蛋白, 发现一新的ASGPR2突变体, 并证实HBxAg与ASGPR2突变体在体外及酵母细胞内均有结合作用.

关键词: N/A

Cloning of a gene coding for novel mutant of asialoglycoprotein receptor 2 binding to hepatitis B virus X protein in hepatocytes

Yin-Ying Lu, Tian-Yan Chen, Jun Cheng, Yao-Dong Liang, Lin Wang, Yan Liu, Jian Zhang, Qing Shao, Ke Li, Ling-Xia Zhang

Yin-Ying Lu, Tian-Yan Chen, Jun Cheng, Yao-Dong Liang, Lin Wang, Yan Liu, Jian Zhang, Qing Shao, Ke Li, Ling-Xia Zhang, Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuan Zhonglu, Beijing 100039, China

Supported by: Grants from National Natural Science Foundation No. C03011402, No. C30070689, and Returned Scholarship of General Logistics Department of PLA.

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

Received: March 8, 2003
Revised: March 20, 2003
Accepted: April 16, 2003
Published online: August 15, 2003

Abstract

AIM

The pathogenesis of HBV-induced malignant transformation is incompletely understood. The X protein of hepatitis B virus (HBxAg) is a multifunctional protein that can influence a variety of signal transduction pathways within the cell and is essential for establishing natural viral infection, it also has been implicated in the development of liver cancer associated with chronic infection. Further understanding of the interaction between HBxAg and proteins in hepatocytes is of great significance for the prevention of the development of hepatocellular carcinoma (HCC).

METHODS

HBxAg bait plasmid was constructed by ligating the HBxAg gene with a yeast expression vector pGBKT7, then transformed into yeast AH109 (a type). The transformed yeast cells were amplified and mated with yeast cells Y187(α type) containing liver cDNA library plasmid pCAT2 in 2×YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection twice. Plasmid of true positive blue colonies was extracted and analysed by DNA sequencing and blast in GenBank. After the complete sequence of the novel mutant of asialoglycoprotein receptor 2 (ASGPR2) was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7 vector, the recombined plasmid was translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro together with HBxAg.

RESULTS

Eighteen genes in forty-one positive colonies were obtained, one of them is a novel mutant of ASGPR2, which is 80 % homologous to natural ASGPR2. The complete sequence of the mutant was amplified from the mRNA of HepG2 cell by RT-PCR successfully. The interaction between HBx and ASGPR2 mutant was further confirmed by immunoprecipitation technique.

CONCLUSION

Interaction between HBx and ASGPR2 mutant can be observed in both yeast cell and in vitro.

Key Words: N/A

0 引言

乙型肝炎病毒(HBV)慢性感染是引发肝细胞癌的主要危险因子, HBV编码的X蛋白(HBxAg)具有潜在的致癌作用, 在多种基因发生异常转化的过程中起转录协同激活作用, 但具体机制不清[1-4]. 许多研究提示HBV X蛋白能抑制肿瘤抑制蛋白p53的功能并影响多种信号转导途径, 对多种增强子及启动子有反式激活作用[5-7]; 还可以抑制肝细胞DNA的损伤修复, 激活细胞信号级联包括促分裂原蛋白激酶(MAPK), Janus家族酪氨酸激酶(JAK)/信号转导子和转录通路激活因子(STAT), 对肝细胞的生长繁殖、细胞凋亡和细胞生长检测点的调节有着广泛的影响, 在HBV诱导肝细胞癌的发生中起着重要作用[8-10]. 筛选肝细胞中与HBxAg的结合蛋白对探讨HBV致癌的细胞效应机制提供重要线索.

1 材料和方法

1.1 材料

AH109酵母菌株(MA Ta, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4△, gal80△, LYS2: : GAL1_UAS-GAL1_TATA-HIS3, GAL2_UAS-GAL2_TATA-ADE2URA3: : MEL1 _TATA-lac Z MEL1)、预转化的cDNA肝文库(Y187)、pGBKT7-BD克隆载体及酵母YPD培养基、SD/-Trp、SD/-Leu, SD/-Trp-Leu-His, SD/-Trp-Leu-His-Ade等培养基、X-α-gal购于Clontech公司. 大肠杆菌DH5α及HBV ayw亚型基因全序列质粒载体pCP10、HepG2细胞为本室保存, c-myc单克隆抗体本室自制. 辣根过氧化物酶标记羊抗鼠IgG购于北京中山生物公司. Taq DNA 聚合酶、T4 DNA连接酶、EcoRI和PstI、BamH I购于Takara生物公司. 丙烯酰胺、N, N'-亚甲双丙烯酰胺、IPTG及X-β-Gal及pGEM-T载体、Trizol总RNA提取试剂盒、RT-PCR试剂盒购于Promega公司. TEMED购于宝林曼公司. 醋酸锂、半硫酸腺苷购于Sigma公司. HBxAg基因扩增引物(P1 5'-GAATTCATGGCTGCTAGGCTGTGCTG-3'和P2 5'-CTGCAGATGGTGCTGGTGCG CAGACC-3'), 肝文库插入基因序列扩增引物(P3 5'-CTATTCGATGATGAAGATACCCCACCAAACCC-3', P4 5'-GTGAACTTGCGG GGTTTTTCAGTATCTACGA-3'), 去唾液酸蛋白受体突变体基因扩增引物(P5 5'-GAATTCATGGCCAAGGACTTTCAAGATATCC-3', P6 5'-GGATCCTCAGGCC ACCTCGCCGGTGGCATTC-3')合成及DNA测序由上海博亚公司承担.

1.2 方法

1.2.1 诱饵质粒的构建及表达 PCR扩增HBxAg基因与pGBKT7载体连接, 酶切鉴定后醋酸锂法将诱饵质粒转化入酵母AH109株后在SD/-Trp培养基上筛选生长6-8 d, 挑取阳性菌落培养后提酵母蛋白质, 用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western免疫印迹法验证HBxAg在酵母中的表达.

1.2.2 诱饵与肝文库的酵母配合 挑取在SD/-Trp培养基上生长的pGBKT7-HBxAg质粒的酵母AH109菌落一到数个接种于SD/-Trp培养基中, 30 °C 250 r.min^-1振摇过夜, 次日离心后用2×YPDA培养液5 mL重悬细胞, 计数细胞数大于1×10¹².l^-1时与1 μL的肝文库酵母细胞在50 mL 2×YPDA中30 °C 30-50 r.min^-1配合18-24 h, 离心用1×YPDA 10 μL重悬细胞, 分别取250 μL铺于15 cm的SD/-Trp-Leu-His(3缺), SD/-Trp-Leu-His-Ade (4缺)培养基各25块上, 同时将配合产物按1:10、1:100、1:1000铺于SD/-Trp-Leu培养基上检验配合效率. 生长6-18 d后挑取大于直径3 mm的菌落再次画线于铺有X-α-gal的4缺培养基上检查X-α-gal酶活性, 在此培养基上能生长且变成蓝色的为真阳性菌落. PCR扩增出目的片段后测序并进行生物信息学分析.

1.2.3 新基因全序的克隆及重组表达载体的构建 根据GenBank的序列信息设计引物P5、P6, 以HepG2细胞的mRNA为模板, 逆转录PCR(RT-PCR)扩增ASGPR2基因的全序列, 送DNA测序鉴定后EcoR I、BamH I双酶切克隆入酵母表达载体pGADT7.

1.2.4 体外免疫共沉淀 TNT网织红细胞裂解物体外翻译HBxAg和ASGPR2突变体(在此过程中掺入35S), 二者各取5 μl在1.5 μL的微离心管中冰上混合, 30 °C孵育1 h, 加入470 μL免疫共沉淀缓冲液、10 μL G蛋白-琼脂糖珠、10 μL c-myc单克隆抗体、4 °C孵育2 h. 14000 r.min^-1. min^-1离心1-2 min, 弃上清. 加入TBST 0.5 mL洗3次, 加15 μL SDS上样缓冲液, 80 °C加热变性5 min, 瞬时离心后将上清10 μL上样到SDS-PAGE胶上电泳, 清洗固定凝胶后, 恒定60 °C真空干燥40 min, -20 °C条件下放射自显影.

2 结果

2.1 pGBKT7-HBx重组诱饵质粒的构建及表达

扩增出的HBxAg基因连接到pGBKT7载体中经酶切鉴定结果正确. 转化入酵母AH109株筛选到阳性菌落, 提酵母蛋白质进行SDS-PAGE和Western免疫印迹分析, 结果显示对照无表达而转化了pGBKT7-HBx的酵母蛋白提取物Western印迹分析可见明显目的条带, 说明HBxAg基因已成功地在酵母中表达.

2.2 诱饵与肝文库酵母菌株配合结果

配合后筛选出既能在四缺( SD/-Trp-Leu-Ade-His)培养基又能在铺有X-α-半乳糖(X-α-gal)的四缺培养基上均能生长并变成蓝色的真阳性菌落41个, 用文库扩增引物PCR扩增出目的片段并测序, 结果在GenBank中进行分析, 发现其中1个含ASGPR2突变体基因.

2.3 新基因全序的克隆及重组结果

用引物P5、P6从HepG2细胞的mRNA中逆转录扩增出ASGPR2突变体基因的全序列(图1, 图2), 测序鉴定正确后克隆入酵母表达载体pGADT7, 酶切鉴定结果正确(图3).

图1 ASGPR突变体基因序列图(GenBank号: AF 529374).

图2 新的去唾液酸蛋白受体突变体与GenBank中去唾液酸蛋白受体2的DNA序列比较. G: GenBank的序列; M: 受体突变体序列.

图3 pGADT7-ASGPR质粒酶切鉴定. 1: DNA Marker; 2: pGADT7-ASGPR质粒EcoR I/Ps tI双酶切.

2.4 体外免疫共沉淀结果

放射自显影照片结果显示, 在泳道2出现各自的两条带的分子量大小正确, 说明HBxAg与该去唾液酸蛋白受体新的突变体在体外也能结合(图4).

图4 体外免疫共沉淀图. 1: HBxAg; 2: HBxAg+ASGPR.

3 讨论

HBV慢性感染可导致肝硬化、肝细胞癌发生, 其中HBxAg在致瘤方面的作用尤为重要, 已有研究证实HBxAg引起细胞基因异常是导致肝细胞癌过程中肝细胞异常增生的早期事件, 但具体机制尚未清楚. 近年来发现HBxAg的功能非常复杂[11], 他能通过不同途径 (包括刺激蛋白激酶活性) 影响信号转导通路、反式激活各种细胞及病毒的启动子及增强子、抑制p53蛋白作用、妨碍DNA损伤修复, 并能通过诱导或封闭细胞凋亡来协调细胞增生及程序性坏死间的平衡[12-19], 从而引起肝细胞基因突变, 肝细胞癌的发生.持续低水平的HBxAg表达可导致HCC的发生, 但HBxAg的结构中不含能提供其细胞定位及在病毒感染肝细胞中起作用的特定的核定位信号、DNA结合位点及其他的蛋白质结构域. 明确HBxAg在肝细胞中到底与哪些蛋白质因子相互作用、如何作用, 对于研究HBV在自然感染中所起的具体生物学功能是非常关键的[20-26].

酵母双杂交系统是新近发展起来的一种分子生物学技术, 能有效分析真核细胞中蛋白-蛋白、蛋白-DNA、蛋白-RNA相互作用. 该系统通过将两个推定相互作的用蛋白X和Y被分别融合到一酵母转录激活因子的BD和AD上, X与Y的相互作用重构了激活因子的完整性能导致下游"报告基因"的转录, 产生易探测到的表型, 间接证实X与Y蛋白间的结合作用[27-30]. 利用Clontech公司新推出的酵母双杂交系统3, 我们在真核表达载体pGBKT7中构建pGBKT7-HBx诱饵质粒并在酵母菌株AH109中表达了HBxAg 基因, 与人肝cDNA文库的酵母菌株Y187进行配合, 筛选出与之相互作用的蛋白基因18种, 其中一个基因与ASGPR2的基因高度同源, 推测是一个新的突变体, 该突变体在第68-69位碱基间比ASGPR2基因多57个碱基, 在244-248位碱基间较ASGPR2基因少15个碱基.

体内糖蛋白通过去唾液酸糖蛋白途径代谢, 他们被去唾液酸后通过去唾液酸糖蛋白受体在肝脏被吸收.由于肝细胞表面富含ASGPR, ASGPR现已被用做多种抗-HBV药物的肝细胞靶向载体, 以增强抗肝炎病毒药物的疗效[31]. 有报道从患者体内分离出的HBV病毒粒子能与ASGPR结合, 进一步研究发现HBV是通过前S1相关的包膜结合位点与ASGPR结合, 介导肝细胞对HBV病毒粒子的胞饮作用, 可能是HBV进入肝细胞的一个机制[32,33]. 在本项研究中发现HBxAg能与ASGPR2结合, 对于研究HBxAg在HBV感染及HCC发生中的具体功能又提出了新的线索, 二者结合后会引起哪些的生物学效应是下一步研究的重点.

备注

$[Footnotes]

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