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在用10~(-5)mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.
After 10~(-5) mol/L all-trans retinoic acid (RA) was used to induce the differentiation of human lung adenocarcinoma cell line GLC-82, the M13 phagemid pSPORT1 was used as a vector, and directional cloning was used to construct them. Three cDNA libraries without RA-induced and RA-induced 1d and 4d cells were used for subtractive hybridization and enrichment of target-sense single-stranded DNA containing recombinants and uninduced library cDNA driver. RA-specific single-stranded DNA was used to restore the enriched single-stranded DNA to double-stranded and transform competent bacteria. A cDNA subtracted library of activated expression genes was established during cell differentiation, and 124 cDNA subtracted clones were obtained. Southern blotting was performed with the total cDNA of the library, and the RNA of the cells before and after RA induction was subjected to Northern blot hybridization. Two (RA5, RA28) were screened for early transient expression and one (RA42) early and continuous expression. The cDNA clones were full-length cDNAs of 1.8, 1.5 and 0.7 kb. Sequence analysis and preliminary functional analysis showed that the three first reported sequences of RA5, RA28 and RA42 may be cDNA clones of human lung adenocarcinoma cell differentiation-related genes. .