Xu Jin-song, Cai Shao-xi, Zou Fei, Tong Wan-cheng, Wan Wei-ren, Zhao Hai-jin
Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Jan;26(1):82-5.
To construct a subtracted cDNA library of differentially expressed genes in eosinophils from asthma patients.
Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the eosinophils of asthma patients before and after treatment. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library, followed by amplification of the library through E. coli transformation with calcium chloride and screening of blue and white clones of the transformants. One hundred positive bacterial clones were randomly picked and identified by colony PCR.
The amplified library contained more than 3,000 positive bacterial clones. Analysis of the randomly selected 100 white clones by PCR showed that 90% of the clones contained 100-500 bp inserts, which might be the cDNA fragments of differentially expressed genes in eosinophils of asthma patients before treatment.
A subtracted cDNA library of differentially expressed genes in the eosinophils of asthma patients before and after treatment is constructed successfully by SSH and T/A cloning techniques, which lays a solid foundation for screening and cloning new specific differentially.expressed genes in the eosinophils of asthma patients.
构建哮喘患者嗜酸性粒细胞中差异表达基因的消减cDNA文库。
采用抑制性消减杂交（SSH）技术分离哮喘患者治疗前后嗜酸性粒细胞中差异表达基因的cDNA片段。将这些cDNA片段直接插入T/A克隆载体以建立消减文库，随后通过氯化钙转化大肠杆菌对文库进行扩增，并筛选转化子的蓝白克隆。随机挑选100个阳性细菌克隆，通过菌落PCR进行鉴定。
扩增后的文库包含3000多个阳性细菌克隆。对随机选取的100个白色克隆进行PCR分析表明，90%的克隆含有100 - 500 bp的插入片段，这些片段可能是哮喘患者治疗前嗜酸性粒细胞中差异表达基因的cDNA片段。
利用SSH和T/A克隆技术成功构建了哮喘患者治疗前后嗜酸性粒细胞中差异表达基因的消减cDNA文库，为筛选和克隆哮喘患者嗜酸性粒细胞中新的特异性差异表达基因奠定了坚实基础。
