南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (01): 82-85.

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应用抑制消减杂交技术构建哮喘病人嗜酸细胞差异表达基因消减cDNA文库

徐劲松;蔡绍曦;邹飞;佟万成;万为人;赵海金;   

  1. 南方医科大学南方医院呼吸科； 南方医科大学热带卫生医学系； 南方医科大学南方医院呼吸科 广东 广州 510515； 广东 广州 510515；
  • 出版日期:2006-01-20 发布日期:2006-01-20
  • 基金资助:
    国家自然科学基金(30270593);广东省自然科学基金(A001031)

Construction of subtracted cDNA library by suppression subtractive hybridization for differ- entially expressed genes in eosinophils from asthma patients

XU Jin-song, CAI Shao-xi, ZOU Fei, TONG WAN-cheng, WAN Wei-ren, ZHAO Hai-jin Department of Respiratory Diseases, Nanfang Hospital, Department of Tropical Hygiene, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学南方医院呼吸科； 南方医科大学热带卫生医学系； 南方医科大学南方医院呼吸科 广东 广州 510515； 广东 广州 510515；
  • Online:2006-01-20 Published:2006-01-20

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摘要： 目的构建哮喘病人治疗前后嗜酸细胞差异表达基因的消减cDNA文库。方法采用新近建立的抑制消减杂交方法,哮喘治疗前后的嗜酸细胞为试验和对比材料,分离哮喘病人嗜酸细胞中差异表达基因的cDNA片段,将其与T载体进行T／A连接构建文库,将连接产物用氯化钙转化法转化大肠杆菌进行文库扩增和蓝白斑筛选,随机挑取100个白色克隆用菌落．PCR进行鉴定。结果扩增消减cDNA文库获得3 000余个白色阳性克隆,随机挑取100个白色克隆用 PCR进行扩增,90％的克隆中均有200-600 bp的插入片段,这些片段可能是哮喘嗜酸细胞差异表达基因的cDNA片段。结论用SSH法及T／A克隆技术成功构建了哮喘病人治疗前后嗜酸细胞差异表达基因消减cDNA文库．该消减 cDNA文库的建立为进一步筛选、克隆哮喘病人嗜酸细胞差异表达的新基因奠定了基础。 更多还原

Abstract: Objective To construct a subtracted cDNA library of differentially expressed genes in eosinophifs from asthma patients. Methods 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. Results 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. Conclusion 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.

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