乙型肝炎病毒前-S2蛋白结合蛋白基因S2-29的克隆化研究

摘要

目的

筛选人肝细胞中与乙型肝炎病毒(HBV)前-S2蛋白(Pre-S2)相互作用的蛋白, 探寻HBV致病机制.

方法

用多聚酶链反应 (PCR)技术扩增HBV前-S2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, 用营养缺陷型培养基及蓝白斑双重筛选阳性菌落, 提取酵母质粒转化大肠杆菌并测序, 进行生物信息学分析. 逆转录多聚酶链反应(RT-PCR)方法从HepG2细胞的mRNA中扩增出完整的新基因S2-29, 连入另一酵母表达载体pGADT7, 并用免疫共沉淀方法再次证实二者间的相互作用.

结果

成功克隆出HBV前-S2基因并在酵母细胞中表达, 配合后筛选出既能在四缺(SD/-Trp-Leu-His-Ade)培养基又能在铺有X-α-半乳糖(X-α-gal)的四缺培养基上生长并变成蓝色的真阳性菌落26个, 其中有1个未知基因S2-29, 该基因能在HepG2细胞中表达, 免疫共沉淀方法证实二者在体外也有结合作用.

结论

成功克隆出与乙型肝炎病毒前-S2蛋白相结合的新基因, 为进一步研究HBV前-S2的作用提供了新线索.

关键词: N/A

Screening and identification of a novel gene coding for hepatitis B virus pre-S2 antigen interacting protein S2-29

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

Yin-Ying Lu, Tian-Yan Cheng, Jun Cheng, Yao-Dong Liang, Lin Wang, Yan Liu, Ke Li, Jian Zhang, Qing Shao, 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, Professor, 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 Pre-S2 region of hepatitis B virus (HBV) has been reported to have complex biological functions. It has human polymerized albumin receptor (PAR) activity, which correlates with viral replication, and it can induce neutralization antibody. As an important part of truncated middle surface proteins (MHBs), the Pre-S2 domain binds PKC alpha/beta and triggers a PKC-dependent activation of the c-Raf-1/MAP2-kinase signal transduction cascade, resulting in activation of transcription factors such as AP-1 and NF-kB. To investigate the biological function of hepatitis B virus (HBV) Pre-S2 protein, we used yeast two-hybrid technique to screen proteins interacting with HBV Pre-S2 antigen in hepatocytes.

METHODS

The HBV Pre-S2 gene was amplified by polymerase chain reaction (PCR) and cloned into yeast expression vector PGBKT7 to construct HBV Pre-S2 bait plasmid. The bait plasmid was transformed into yeast AH109 and mated with yeast Y187 containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was 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 and screening. After being extracted and sequenced, genes were analyzed by bioinformatics. The complete sequence of new gene S2-29 was amplified from the mRNA of HepG2 cell by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7, then translated by using reticulocyte lysate and analysed by immunoprecipitation technique in vitro.

RESULTS

Twenty-six colonies were obtained, among them two colonies were new genes with unknown function and no homeobox genes were found in Genbank by blast. The complete sequence of new gene S2-29 could be amplified from the mRNA of HepG2 cell and the interaction between HBV Pre-S2 antigen and S2-29 was further confirmed by coimmunoprecipitation technique.

CONCLUSION

Genes of HBV Pre-S2 interacting proteins were successfully screened. A novel gene S2-29 was cloned and could express in HepG2 cell. The HBV Pre-S2 antigen could interact with S2-29, which brings new clues for studying the biological functions of HBV Pre-S2 and the pathogenesis of HBV infection.

Key Words: N/A

0 引言

乙型肝炎病毒(HBV)的前-S2(Pre-S2)蛋白是HBV外膜蛋白的组成部分之一, 因有多聚人血清白蛋白(PHSA)结合位点而与HBV的嗜肝性及入侵肝细胞有关[1-3]; 前-S2蛋白上存在B淋巴细胞识别位点, 有较强的免疫原性, 可诱导中和抗体的产生, 在HBV疫苗的研究和应用方面深受重视[4-10]; 近年来还发现HBV前-S2区域是HBV截短型中蛋白的反式激活功能的重要功能域, 与肝脏肿瘤发生密切相关[11,12], 使前-S2蛋白的功能变得更加复杂, 明确其具体的作用机制并进行相应的阻断对于HBV感染的防治有着重要的意义; 我们用酵母双杂交技术筛选其结合蛋白基因, 找到肝细胞能与前-S2蛋白结合的新基因S2-29, 为研究前-S2蛋白的生物学功提供新的线索.

1 材料和方法

1.1 材料及主要试剂

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为本室保存, 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购于北京中山生物公司. Taq DNA 聚合酶、T4 DNA连接酶、BamHI和PstI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. 多聚酶链反应技术扩增Pre-S2基因片段所用引物(P1 5'- GGATCCATGCAGTGGAATTCCACAACCTTCC-3', P2 5'-CTGCAGGTTCAGCGC AGGGTCCCCAATCCTC-3'), 根据表达载体外源基因插入位点设计肝cDNA文库插入序列扩增引物(P3 5'-CTATTC GATGATGAAGATACCCCACCAAACCC-3', P4 5'-GTGAACTTGCGGGGTTTT TCAGTATCTACG A-3'), 未知基因S2-29扩增引物(p5 5'-GAATTCATGGGGGCCACTGAGGTTGGGGATG-3', p6 5'-GGATCCTCACCCCCCTCTTCTTCTGATGTGG -3')合成及DNA测序在上海博亚公司进行.

1.2 方法

1.2.1 诱饵质粒的构建及表达 PCR从HBV ayw亚型基因全序列质粒载体pCP10中扩增HBV前-S2基因, 与pGBK-T7载体连接, 酶切鉴定后转化入酵母菌株AH109, 提取酵母蛋白质, 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HBV 前-S2基因在酵母中的表达.

1.2.2 诱饵与肝文库的酵母配合 pGBKT7载体中有色氨酸(Trp)基因, 转化成功的酵母菌株能在缺乏Trp的SD平板上生长, 挑取在SD/-Trp培养基上生长的pGBKT7-HBV前-S2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 mL的肝文库酵母细胞(含有亮氨酸Leu 基因)在50ml 2×YPDA中30 °C 30-50r.min^-1配合18-24 h, 离心, 用1×YPDA 10 mL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 未知基因完整序列的克隆及测序 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RT-PCR扩增S2-29基因的全序列, EcoRI、BamH I双酶切克隆入酵母表达载体pGADT7, 鉴定后送DNA测序.

1.2.4 体外免疫共沉淀再次证实结合作用 TNT网织红细胞裂解物体外翻译HBV Pre-S2 Ag和S2-29蛋白(在此过程中掺入³⁵S), 二者各取5 μL在1.5 mL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蛋白-G琼脂糖珠、10 μL c-myc单克隆抗体、4 °C孵育2 h. 14000 r.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-HBV Pre-S2重组诱饵质粒的构建及表达

利用自行设计的引物P1、P2成功扩增出HBV 前-S2蛋白基因片段, PCR产物经10 g/L琼脂糖凝胶电泳分析显示扩增片段约165 bp, 与预期片段符合, 且无非特异扩增现象, 测序结果显示完全符合报告序列. 用BamH I及Pst I双酶切所得片段, 连接到用相同酶所切的pGBKT7载体中, 经酶切鉴定结果正确. 用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, 挑取阳性菌落培养并提酵母蛋白质, 进行SDS-PAGE和Western免疫印迹分析. 结果显示转化了pGBKT7- HBV前-S2的酵母蛋白提取物Western印迹分析可见明显目的条带(约24 kd, 其中c-myc 1.3 kD, DNA结合域17 kD, 前-S2蛋白约6 kD), 且无杂带, 说明HBV 前-S2蛋白基因已成功地在酵母中表达(图1).

图1 Western免疫印迹图. 1: (pGBKT7-HBV PreS2)酵母蛋白提取物; 2: 蛋白Marker.

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-His-Ade)培养基生长又能分解X-α-gal变成蓝色的真阳性菌落26个, PCR扩增出目的片段后, 测序结果在GenBank中进行分析, 发现其中有未知基因2个.

2.3 未知基因完整序列的克隆及测序

RT-PCR方法成功克隆出S2-29新基因的完整序列(图2), 大小为342 bp, GenBank注册号AF 497566, 并连接入pGADT7载体中.

图2 前-S2-29新基因序列(GenBank号: AF 497566).

2.4 体外免疫共沉淀再次证实结合作用

HBV前-S2蛋白(Mr 6000), 新基因编码蛋白的相对分子质量为(Mr 12500), 经体外翻译相互作用SDS电泳后, 放射自显影图上可看出(图3), 1泳道两条带的大小正确, 对照2泳道仅有HBV 前-S2蛋白的一条带, 进一步证实二者在体外也能相互结合.

图3 免疫共沉淀图. 1: HBV PreS2+S2-29; 2: HBV PreS2.

3 讨论

HBV前-S2蛋白作为HBV包膜蛋白的组成之一, 功能复杂, 不仅能与PHSA结合介导肝细胞的黏附及入侵, 在其肽段内还包含有I、II类MHC限制性和T细胞受体(TCR)的不同功能位点和特异性T、B淋巴细胞结合位点, 能引起中和抗体和保护性免疫的发生, 可用于构建新型的乙型肝炎疫苗[13-18]. 近年来还有报道前-S2蛋白N-末端I区的存在对于截短型中蛋白(MHBst)的反式激活作用是必不可少的, MHBst通过启动不同的细胞内信号系统分别来激活转录因子Ap-2、NF-κB、血清应答元件(SRE)以及转录因子Sp1, 从而完成其反式激活功能, 提示前-S2蛋白可能参与HBV慢性化及HCC的发生过程. 弄清HBV前-S2蛋白在肝细胞中到底与那些蛋白质因子相互作用、如何作用, 对于研究HBV在自然感染中的具体过程和机制是非常关键的.

酵母双杂交系统是近年来新发展起来的一种分析真核细胞中蛋白-蛋白、蛋白-DNA、蛋白-RNA相互作用的一种有效的基因分析方法, 他通过将两个推定有相互作用的蛋白X和Y分别融合到一酵母转录激活因子的DNA结合域(BD)和转录激活域(AD)上, X与Y的相互作用使BD和AD重新结合在一起, 重构了激活因子的完整结构功能, 导致下游报告基因的转录、表达[19-23]. 基于该原理, 我们在真核表达载体pGBKT7中构建pGBKT7-HBV 前-S2诱饵质粒并在酵母菌株AH109中表达了HBV前-S2基因, 与人肝cDNA文库的酵母菌株Y187进行配合, 寻找与前-S2蛋白相互作用的蛋白, 经过营养缺陷和蓝白斑双重选择[24-30], 筛选出与HBV前-S2蛋白有相互作用的未知功能的新基因S2-29, 将其在GenBank中进行比较未找到与之同源的基因序列, 用自行设计的引物能从肝癌细胞系HepG2细胞的mRNA中逆转录出该基因的完整序列, 说明该基因在之前从未被报道过, 是一个全新的基因序列, 并能在肝癌细胞系HepG2中表达. 为进一步确证, 我们又用体外免疫共沉淀的方法再次证实二者在体外也有相互作用[31,32], 由此得出结论HBV前-S2蛋白能与肝细胞中的未知基因S2-29表达产物结合, 并可能在HBV感染肝细胞的过程中起着一定作用.

以上结果为前-S2蛋白的功能研究提供了新的有价值的线索, 对HBV致病机制的探讨也提出了全新的主题, 但我们还需要进一步探明其间相互作用的具体机制、作用结果及其对HBV前-S2蛋白生物学功能的影响, 为寻找阻断HBV感染及原发性肝癌(HCC)发生的有效方法开辟新道路.

备注

$[Footnotes]

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