酵母双杂交技术筛选鉴定乙型肝炎病毒e抗原结合蛋白E-19的研究

摘要

目的

乙型肝炎病毒e抗原(HBeAg)对HBV的感染和复制都不是必须的, 普遍认为其与HBV引起免疫耐受、免疫系统功能障碍有关. 筛选并克隆人肝细胞cDNA文库中与HBeAg相互作用蛋白的基因, 明确其具体作用机制.

方法

应用酵母双杂交系统3, 将多聚酶链反应(PCR)法扩增的HBeAg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pACT2的酵母细胞Y187进行配合, 在营养缺陷型培养基和X-α-半乳糖(X-α-gal)上进行双重筛选阳性菌落, 提取阳性酵母菌落的质粒转化大肠杆菌氨苄青霉素-LB平板上选择并测序, 结果在GenBank中进行生物信息学分析. 并根据GenBank中的序列信息设计引物从并克隆到另一酵母表达载体pGADT7中, 体外免疫共沉淀方法再次验证二者之间的结合作用.

结果

成功克隆出HBeAg基因并在酵母细胞中表达, 与肝文库配合后选出既能在四缺(SD/-Trp-Leu-His-Ade)培养基又能使X-α-gal变成蓝色的真阳性菌落39个, 其中有5个未知基因, 在GenBank中未找到同源序列, 在HepG2细胞的mRNA中成功扩增出该基因的全序, 体外免疫共沉淀方法证明HBeAg与新基因E-19表达的蛋白质在体外也有结合作用.

结论

成功克隆出HBeAg的肝细胞结合蛋白, 发现与HBeAg有相互作用的未知蛋白新基因, 为HBeAg的功能研究提出新线索.

关键词: N/A

Screening and identification of a novel hepatitis B virus e antigen binding protein E-19 in hepatocytes by yeast two-hybrid technique

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

Yin-Ying Lu, Qing Shao, Jun Cheng, Tian-Yan Cheng, Lin Wang, Yao-Dong Liang, Yan Liu, Jian Zhang, 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 hepatitis B precore Ag (HBeAg) is a secreted nonparticulate version of the viral nucleocapsid hepatitis B core Ag (HBcAg), and its function is unknown. Some researchers have proposed that the HBeAg may have an immunoregulatory function in promoting viral persistence and is associated with immunologic tolerance. To investigate biological functions of hepatitis B virus e protein(HBeAg), yeast two-hybrid technique was employed to seek proteins in hepatocytes interacting with HBeAg.

METHODS

HBeAg bait plasmid was constructed by ligating HBeAg gene with 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 were extracted and analysed by DNA sequencing and blast in GenBank. After the complete sequence of new gene E-19 was amplified from the mRNA of HepG2 cell by 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 HBeAg.

RESULTS

Twenty genes in thirty nine positive colonies were obtained, there were five new genes.The complete sequence of new gene E-19 was successfully amplified from the mRNA of HepG2 cell by RT-PCR. The interaction between HBeAg and expression products of new gene E-19 was further confirmed by immunoprecipitation technique.

CONCLUSION

Genes of HBeAg interacting proteins in hepatocytes were successfully cloned and HBeAg could bind with the protein expressed by new gene E-19.

Key Words: N/A

0 引言

乙型肝炎病毒e抗原(HBeAg)是核壳蛋白的分泌型, 研究发现HBeAg对乙型肝炎病毒(HBV)的感染和复制无重要作用, 有学者提出他可能具有免疫调节的作用而导致HBV感染的持续[1-3]. HBeAg可阻断细胞毒性T淋巴细胞优先清除对HBcAg特异的Th1细胞及Th1细胞介导的抗-HBc抗体反应, 使免疫应答转换为Th2细胞亚型, 使HBV逃避免疫清除, 感染慢性化[4-7]. 但HBeAg与肝细胞之间的相互作用的机制尚不清楚, 此方面的研究较少, 从寻找HBeAg与肝细胞间的相互作用蛋白入手, 可望在研究HBeAg的确切生物学功能方面有所发现[8-10].

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、pGADT7-AD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, 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、EcoR I和Pst 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、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. HepG2细胞购于中科院上海细胞生物研究所. HBeAg扩增引物(P1 5'- GAATTCATGCAACTTTTTCACCTCTG-3', P2 5'-CTGCAGGCCCCAAAGCCACCCAAGGC-3', 新基因E-19扩增引物(P3 5'- GAATTCATGTCATGGACACCCACCTC-3', P4 5'-GGATCCAGAAAGAAACA GGG TGAGGG -3'的合成及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HBeAg基因与pGBKT7载体连接, 酶切鉴定后醋酸锂法转化入酵母菌株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HBeAg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-HBeAg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的肝文库酵母细胞在50 mL 2×YPDA中30 °C 30-50 r.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.提取阳性酵母细胞中的质粒, 电穿孔法转化大肠杆菌, 氨苄青霉素平板筛选阳性克隆并测序.

1.2.3 新基因全序及表达载体的克隆及重组 根据GenBank的序列信息设计引物P3、P4, 以HepG2细胞的 mRNA为模板, 逆转录RT-PCR扩增E-19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HBeAg和E-19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 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-HBeAg重组诱饵质粒的构建及表达

成功扩增出HBeAg基因片段, 连接到pGBKT7载体中酶切鉴定结果正确. 诱饵质粒转化酵母AH109株, 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-HBeAg的酵母蛋白提取物Western印迹分析可见明显目的条带(图1).

图1 pGBKT7-HBeAg酵母表达Western免疫印迹分析.

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)的四缺培养基上生长并变成蓝色的真阳性菌落39个, 其中有5个未知基因, 在GenBank中未找到同源序列.

2.3 新基因全序及表达载体的克隆及重组结果

RT-PCR方法成功克隆出E-19新基因的完整序列(图2), 大小为351 bp, GenBank注册号AF529373, 并连接入pGADT7载体.

图2 E-19新基因序列(GenBank号AF 529373).

2.4 体外免疫共沉淀再次证实结合作用

HbeAg的Mr 25000, 新基因E-19编码蛋白的Mr 13000, 经体外翻译相互作用SDS电泳后, 放射自显影图上可看出, 1泳道两条带的大小正确, 对照2泳道仅有HBeAg一条带, 证实二者在体外也能相互结合(图3).

图3 免疫共沉淀结果. 1: HBeAg+E-19; 2: HBeAg.

3 讨论

近年来由于乙肝疫苗的广泛应用对HBV感染起到了一定的阻断作用, 但全世界仍有超过3.5亿人处于HBV慢性感染状态, 成为重要的传染源, 其中有很大部分要转化为肝硬化、肝癌[11-14]. 在HBV感染的漫长过程中, 大多数感染者由于HBeAg的阴转及抗-HBe的出现而伴随病毒复制明显减少; 但少数HBeAg阴性的感染者体内病毒仍有持续或间断的高水平复制, 并伴肝细胞坏死性炎症及进行性纤维化. 既往的研究结果普遍认为HBeAg是一种免疫调节因子, 可调节宿主的免疫应答, 抑制宿主T细胞的细胞毒活性, 形成对HBV感染的免疫耐受性[15-17]. 另一方面, 由于HBeAg含有病毒高度保守的体液和细胞抗原靶位, 与HBcAg有部分共同的序列, 有相同的抗原靶位, 当HBeAg发生突变时, 失去血清中HBeAg的调节, 表达HBcAg的肝细胞经受增强的T细胞的细胞毒作用, 可导致病情加重[18-22]. 血中抗-HBe长期阳性的患者, 发生肝硬化、肝癌的几率较高, 原因不清, 这些发现使得HBeAg的生物学功能变得更复杂. 寻找肝细胞中HBeAg的相互作用蛋白, 并进一步探明其机制, 对于明确上述问题有着重要意义.

在本项研究中所使用的酵母双杂交系统3, 在原有系统的基础上增加了报告基因的数量, 分别与BD和AD结合的HBeAg和肝文库中的蛋白要确实有相互结合作用, 才能同时激活下游的3个报告基因, 表达3种编码产物, 加上2个载体上各自带有的氨基酸报告基因, 筛出的真阳性菌落才能在缺乏4种氨基酸的培养基上生长, 并能分解x-α-gal显现蓝色. 另外, 该系统利用a型和α型酵母配合形成的二倍体细胞内, 诱饵质粒与文库质粒所表达的蛋白质可以相互作用的原理, 免去了需要共转染2种质粒所带来的低效率问题, 并将真阳性率提高到95%, 大大增强了结果的可靠性[23,24].

我们应用此技术构建了pGBKT7-HBeAg诱饵表达质粒, 在预转化的人肝cDNA文库中"钓"出与HBeAg有相互作用的蛋白基因20种, 其中3种为未知基因, 在GenBank中未发现与之同源的表达基因序列[25-31]. 根据GenBank的信息, 我们自行设计了新基因E-19的上下游引物, 并成功地从HepG2细胞的mRNA中逆转录出该基因的完整序列, 说明该基因能在HepG2细胞中表达. 将其连入另一酵母表达载体pGADT7, 与pGBKT7-HBeAg一起分别在TNT网织红细胞系统翻译, 掺入同位素, 免疫共沉淀方法分析, 结果显示新基因E-19的表达产物在体外能与HBeAg结合, 聚丙烯酰胺凝胶电泳可见二者配合后的两条带, 对应的分子量大小正确. HBeAg的肝细胞结合蛋白新基因E-19的发现及证实, 提示我们对HBeAg生物学功能、HBV致病机制的认识存在很大的局限性, 目前的研究着眼点可能遗漏了很多重要的方面, 或许有更关键的因素没有被发现, 而使研究工作误入歧途; 新基因的发现同时也为今后的科研工作指明了新的方向.

备注

$[Footnotes]

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