应用白细胞cDNA文库的酵母双杂交技术筛选HCTP4结合蛋白基因

摘要

目的: 用酵母双杂交技术筛选白细胞中与HCTP4蛋白结合的蛋白的编码基因.

方法: 用多聚酶链反应(PCR)法扩增HCTP4基因, 连接入酵母表达载体pGBKT-7中构建诱饵质粒, 转化酵母细胞AH109并在其内表达, 然后与转化了人白细胞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, 增菌后提出质粒, 转化入大肠杆菌(DH5), 提出质粒并测序, 进行生物信息学分析.

结果: 成功克隆出HCTP4基因并在酵母细胞中表达, 配合后选出在四缺(SD/-Trp- Leu-Ade- His)培养基及铺有X--半乳糖(X--gal)的四重缺陷培养基上均能生长并变成蓝色的真阳性菌落44个, 其中25个免疫球蛋白λ轻链; 6个来自RP11-189K21克隆的人DNA序列; 4个来自RP11-507C10克隆的人DNA序列; 2个人BAC克隆RPCI11-75L1; 1个人BAC克隆RP11-21M10; 1个人遍素酶智能弹(MIB); 1个来自RP11-867O8克隆的人DNA基因; 1个来自RP3-509I19克隆的人DNA序列; 1个小核糖核蛋白G; 1个UMP-CMP激酶; 1个新基因.

结论: 成功克隆出HCTP4的结合蛋白, 为进一步研究丙型肝炎病毒(HCV)的作用提供了新线索.

关键词: N/A

Screening of HCTP4 interacting proteins in leukocytes by yeast-two hybrid technique

Min Liu, Jun Cheng, Shu-Lin Zhang, Lin Wang, Qing Shao, Jian Zhang, Yao-Dong Liang,

Min Liu, Jun Cheng, Shu-Lin Zhang, Lin Wang, Qing Shao, Jian Zhang, Yao-Dong Liang, 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 the biological function of HCTP4, yeast-two hybrid was performed to screen proteins interacting with HCTP4 in leukocytes.

METHODS: The HCTP4 gene was amplified by polymerase chain reaction (PCR) and HCTP4 bait plasmid was constructed by using yeast-two hybrid system 3, then the constructed vector was transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing leukocytes 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--gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, we underwent analysis by bioinformatics.

RESULTS: Forty-four colonies were sequenced, among which twenty-five colonies were immunoglobulin lambda light chain, six human DNA sequences from clone RP11-189K21, four human DNA sequences from clone RP11-507C10, two homo sapiens 12p BAC RPCI11-75L1, one homo sapiens BAC clone RP11-21M10, one homo sapiens ubiquitin ligase mind bomb (MIB), one homo sapiens genomic DNA, chromosome 11 clone: RP11-867O8, one human DNA sequence from clone RP3-509I19, one homo sapiens small nuclear ribonucleoprotein polypeptide G, one homo sapiens UMP-CMP kinase (UMP-CMPK), and a new gene.

CONCLUSION: Genes of HCTP4 interacting proteins in leukocytes are successfully cloned and the results bring some new clues for studying the biological functions of HCTP4 and associated proteins.

Key Words: N/A

0 引言

丙型肝炎病毒(HCV)基因组为一线状单股正链RNA病毒, 属黄病毒属. 由编码区、5'-非编码区和3'-非编码区组成. 编码区包括两部分, 即结构区与非结构区. 前者较保守, 后者易发生变异. 结构区分C区和E区, 相应的编码物分别是核心蛋白和囊膜蛋白, 由他们组成病毒颗粒. HCV核心蛋白位于病毒多肽的氨基末端, 被宿主的信号肽酶切割后产生. 丙型肝炎病毒核心蛋白除了作为核壳蛋白具有病毒颗粒组装功能外, 还具有多种调控细胞、病毒基因表达、细胞生长以及免疫调节等功能, 其氨基酸序列高度保守, 临床和实验研究显示HCV核心(core)蛋白与肝硬化、肝细胞癌(HCC)发生发展过程密切相关[1-2]. 为了从不同的角度对HCV核心蛋白的反式调节基因进行验证及研究, 我们应用基因表达谱芯片技术(cDNA microarray)和分子克隆方法对HCV核心蛋白反式调节的靶基因成功地进行了筛选和克隆, 并将其中一新的靶基因命名为HCTP4[3], 为更加广泛深入地研究HCV核心蛋白的反式调节基因HCTP4, 我们用酵母双杂交技术筛选白细胞中与HCTP4结合蛋白基因.

1 材料和方法

1.1 材料

Saccharomyces cerevisiae AH109酵母株、Y187酵母株(K1612-1)、cDNA淋巴文库、以上产品均购自Clontech公司. 酵母YPDA培养基、SD/-Trp培养基SD/-Leu、SD/-Trp/-Leu、SD/-Trp/-Leu/-His、SD/-Trp/-Leu/-His/-Ade, X--半乳糖苷酶(Gal)等购自Clontech公司, 半硫酸腺苷、醋酸锂购自Sigma公司. 复杂高效感受态(FSB), 本室自制. 大肠杆菌(DH5), 本室保存.

1.2 方法

1.2.1 诱饵质粒的构建及表达: HCTP4的酵母表达载体pGBKT7-HCTP4用醋酸锂法转入酵母细胞AH109由本室构建.

1.2.2 酵母淋巴文库的构建: cDNA淋巴文库进行增菌后, 提出质粒, 转化入酵母细胞(Y187), 经文库滴定, 确定文库细胞计数大于1×10⁹细胞/mL.

1.2.3 诱饵与淋巴文库的酵母配合: 挑取在SD/-Trp选择培养基上生长转化子(计数大于1×10⁹细胞/mL)与淋巴文库混合, 30 ℃轻摇配合过夜, 24 h后铺板SD/-Trp/-Leu/-His 25块、SD/-Trp/-Leu/-His/-Ade 25块. 同时进行阳性对照实验及文库滴定. 生长16 d后把长出的大于3 mm的酵母集落, 在铺有 X--半乳糖苷酶的QDO上检查-半乳糖苷酶活性, 认为在QDO培养基上生长且出现蓝色菌落的配合为阳性集落.

1.2.4 阳性质粒的克隆和分析: 挑取真正的阳性集落按照试剂盒提供的操作指南Lyticase法提取酵母质粒. 提取的质粒以复杂冰冻高效感受态方法转化大肠杆菌, 于含有氨苄青霉素的SOB平板培养, 所获得的菌落酶切鉴定后测序. 阳性克隆DNA测序后, 提交GenBank比对, 进行生物信息学分析, 并把所获新的基因存入GenBank数据库.

2 结果

2.1 cDNA测序与同源性分析初步结果

配合后筛选出既能在4重缺陷(SD/-Trp/-Leu/- His/-Ade)培养基又能在铺有X--gal 的4重缺陷培养基上均能生长并变成蓝色的真阳性菌落44个克隆测序, 与GenBank数据库进行初步比较. 其中有1 个克隆为新基因, 其余43个均与已知基因的部分序列高度同源(93-100%), 详细结果见表1.

表1 HCTP4蛋白结合的白细胞中蛋白的类型.

序号	筛选出的目的基因	同源性	相同克隆数
1	免疫球蛋白λ轻链	95%	25
2	来自RP11-189K21克隆的人DNA序列	99%	6
3	来自RP11-507C10克隆的人DNA序列	95%	4
4	人BAC克隆RPCI11-75L1	99%	2
5	人BAC克隆RP11-21M10	96%	1
6	人遍素酶智能弹(MIB)	100%	1
7	来自RP11-867O8克隆的人DNA基因	99%	1
8	来自RP3-509I19克隆的人DNA序列	93%	1
9	小核糖核蛋白G	100%	1
10	UMP-CMP激酶	99%	1
11	新基因	100%	1

3 讨论

研究新基因的结构与功能是目前分子生物学研究领域中最具挑战性的工作之一, 可以从不同角度入手[4-5]. 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. 该方法建立以来, 经过不断的完善和发展, 不但可以检测已知蛋白质之间的相互作用, 更重要的在于发现新的与已知蛋白质相互作用的未知蛋白. 蛋白质与蛋白质的相互作用是很多生命现象的基础[6-9]. 本研究应用的是酵母双杂交系统3 (Clontech公司商品化的双杂交系统), 由于有3个表达基因用来筛选及严格的对照, 其阳性率达95%以上, 假阳性率5%以下.

我们在真核表达载体pGBK-T7中构建pGBKT7-HCTP4诱饵质粒并在酵母菌株AH109中表达了HCTP4基因, 与人淋巴cDNA文库的酵母菌株Y187进行配合. 挑取阳性克隆44个, 筛选出与HCTP4相互作用的蛋白基因11种, 其中免疫球蛋白λ轻链、智能弹(mind bomb, MIB)、小核糖核蛋白G、UMP-CMP激酶与免疫调节、信号转导及能量供给等作用相关.

本研究挑取阳性克隆44个, 其中免疫球蛋白λ轻链25个, 占56.8%. 用抑制性消减杂交法筛选HCV肿瘤相关性基因显示免疫球蛋白轻链与染色体易位及核苷酸修复有关[10]. 高度保守的HCV核心蛋白具有免疫调节功能[11-12], HCV核心蛋白与免疫球蛋白轻链的相互作用在HCV患者慢性化及肿瘤发生中起一定作用. 我们用酵母双杂交法进一步证明了HCV核心蛋白的反式调节基因HCTP4与免疫调节相关.

智能弹(mind bomb, MIB)是一种泛素酶, 是通过Delta有效激活Notch信号的基础. MIB与细胞内Delta区相互作用促进他的内在化和遍在性. 细胞移植研究表明MIB的功能是有效的激活邻近细胞Notch的信号基础[13]. 穿膜蛋白Notch作为信号受体, 穿膜蛋白Delta作为配体介导几个不同组织的细胞分化信号. Notch信号可能是从细胞表面直接传递到细胞核, Notch受体在正常发育和肿瘤发生中均有重要作用[14].

在剪接体装配的早期阶段小核糖核蛋白是必需的, 核糖核蛋白复合物为核前-mRNA剪接提供动力[15]. 核苷单磷酸激酶(UMP/CMP)位于细胞质和细胞核[16], 催化三磷酸腺苷(ATP)末端的磷酸盐转移到UMP或CMP[17]. 动力学分析表明UMP/CMP激酶优先选择用ATP作为磷酸盐供体, 但UMP和CMP作为磷酸盐受体是平等的[18]. Liou et al[19]研究显示UMP/CMP激酶对于细胞核酸合成所需的UDP, CDP和dCDP的形成有重要的作用. Pasti et al[20]报道UMP-CMP激酶在UTP, CTP和dCTP磷酸化作用中具有重要的催化作用. 细胞激酶磷酸化作用在核酸类似物的抗病毒治疗中是必需的. 几种胞苷和脱氧胞苷是重要的抗肿瘤和抗病毒药, 这些药物在发挥治疗作用要求逐步磷酸化作用到他们的三磷酸盐结构. UMP/CMP激酶对于核酸类似物的磷酸化作用已明确. 通过细胞酶磷酸化脱氧胞苷是活化这些物质的首要条件[16,20].

丙型肝炎病毒核心蛋白长191 aa, 是一种多功能蛋白质, 具有调节细胞凋亡[21-23]、脂代谢[24]、转录、免疫呈递等作用[25]. 尽管循环中可见到HCV抗体, 但HCV患者感染仍持续且可进展到肝硬化、肝癌[26]. HCV不但在肝细胞复制, 而且在单核细胞、B细胞和T细胞也可复制[27-32]. HCV感染外周血细胞改变免疫细胞功能, 引起无效的免疫反应在HCV发病机制中有重要作用[33]. HCV核心蛋白具有广泛的反式激活作用[34-35], 与宿主蛋白相互作用, 可能是病毒感染导致肝细胞损伤和肝细胞癌发生、发展的重要原因. 我们用酵母双杂交法筛选与HCTP4相互作用的蛋白, 进一步验证了HCV具有免疫调节、信号转导等多种功能.

备注

编辑: N/A

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