应用酵母双杂交系统筛选与CIKS(151-574)相互作用的蛋白质  

Screening of New Binding Partners of CIKS with Yeast Two-hybrid System

作　　者：黄国锦[1] 张智清[1] 姚立红[1] 陈爱珺[1] 徐春晓[1] 朱宏建[1] 柴映爽[1] 严馨蕊[1] 金冬雁[1] 

机构地区：[1]中国预防医学科学院病毒所病毒基因工程国家重点实验室,北京100052

出　　处：《生物工程学报》2003年第2期190-194,共5页Chinese Journal of Biotechnology

基　　金：国家杰出青年科学基金 (B类 )资助 (No .30 0 2 90 0 1)。~~

摘　　要：CIKS(ConnectiontoIKKandSAPK JNK)是最近发现的细胞蛋白 ,能激活IKK和SAPK JNK。应用酵母双杂交系统 ,将CIKS(15 1 5 74)插入载体pAS2 1作为诱铒 ,筛选人HeLa细胞MATCHMAKERcDNA文库 ,以期为阐明NFκB及JNK活性调控的分子机理提供新的线索。筛选得到 6个阳性AD 文库质粒 ,并用酵母双杂交实验验证了阳性AD 文库质粒与CIKS的相互作用。将阳性AD 文库质粒测序并对测序结果做BLAST分析 ,发现它们分别是RIKENcDNA 473340F0 3,PLAC8,CD2 7BP (Siva 1) ,CDC5L ,SnRNPsmB ,DVL2。CIKS能与这些功能各异的蛋白质相互作用 ,表明CIKS在细胞的多种生理活动中发挥作用。The NF-κB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IκB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-κB activation pathway through phosphorylation of IκB, the inhibitor of NF-κB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-κB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/+ X-α-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp /+ X-α-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp /+ X-α-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/+ X-α-gal)plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by HaeⅢ digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E.coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03,PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB,and DVL2. CDC5L is a key component of the multi-protein complex es

关 键 词：CDNA文库 蛋白质相互作用 酵母双杂交系统 CIKS 细胞蛋白 载体 

分 类 号：Q75[生物学—分子生物学]