类泛素FAT10高表达肝癌细胞株Hep3B酵母双杂交cDNA文库的构建

Construction and characterization of a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B

目的:构建类泛素FAT10高表达肝癌细胞株Hep3B的酵母双杂交用cDNA文库.方法:从肝癌细胞Hep3B中提取总RNA,分离mRNA.利用反转录酶M-MLV与Oligo(dT)AnchorPrimer合成1stStrandcDNA,用E.coliDNAPolymerase与E.coliDNALigase将RNA链置换成DNA链,合成2ndStrandcDNA.将双链cDNA与EcoRⅠAdaptor连接,然后用EcoRⅠ/XhoⅠ进行酶切.使用SpinColumn除去短链cDNA与pGADT7载体连接,转化入E.coliDH10B,建成原始文库.然后对其进行扩增并随机挑取单菌落,酶切鉴定重组子插入片段大小.结果:提取的总RNA降解少且分子完整;RNA纯度高,相对分子质量为400-5000bp;成功合成双链cDNA,均符合建库要求;库容量达到1.03×106克隆,原始文库滴度为2.50×109cfu/L,扩增后的文库滴度为3.60×1012cfu/L.插入片段大小分布为0.5-3.5kb,平均长度约为2.0kb.结论:所构建文库的各项指标均达到要求,为筛选FAT10作用蛋白奠定了重要基础.

AIM：To construct a yeast two-hybrid cDNA library from a FAT10-overexpressing human hepatic carcinoma cell line Hep3B.METHODS：Total RNA was prepared from Hep3B cells and used to purify poly（A） mRNA.Double-stranded cDNA was synthesized from the purified mRNA,ligated to EcoR I adaptor,digested with EcoR I/Xho I enzymes,and then cloned into the pGADT7 vector.The recombinant vector was transformed into E.coli DH10B to obtain a primary cDNA library.The primary library was amplif ied and used to determine the size of cDNA inserts through enzyme digestion.RESULTS：The primary cDNA library contained 1.03 × 106 independent clones.The titer of the cDNA library was estimated to be 2.50 × 106 cfu/mL,and that of the amplified library was 3.60 × 109 cfu/mL.The size of the inserts varied from 0.5 to 3.5 kb,with an average value of about 2.0 kb.CONCLUSION：A yeast two-hybrid cDNA library has been successfully generated from FAT10-overexpressing Hep3B cells and can be used for future screening of proteins interacting with FAT10.