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目的 克隆和研究支气管哮喘患儿白细胞介素 4 (IL 4 )近侧启动子区DNA序列的多态性。方法 取 4 0名有明显家族及过敏史的哮喘患儿和 10名正常儿童 ,多聚酶链式反应 (PCR)结合单链构象多态性(SSCP )扩增、筛选IL 4近侧启动子片段 ,进一步构建正常对照和异常条带PCR产物的重组质粒pIL 4 Jx2 ,并用双脱氧链终止法对重组质粒进行序列测定。结果 在 4 0名患儿SSCP分析中发现了 7组异常条带。DNA测序结果显示有四处变异位点位于已知的调控元件之内或毗邻位点 :一名病人 - 2 2 9位C被A所替代 ,该变异恰好位于IL 4正性调节元件 Ⅰ (PRE Ⅰ )元件之内 ;两名病人负性调节元件 Ⅱ (NRE Ⅱ )元件毗邻C被T所替代 ,TATA框前增加一个G ;一名病人STAT 6元件附近缺少了ATTTT五碱基核苷酸。结论 过敏性哮喘患儿IL 4近侧启动子区DNA序列存在多态性 ,这可能与IL 4基因异常表达及哮喘的发病有关。
Objective To clone and study the polymorphism of DNA sequence in the proximal promoter region of interleukin 4 (IL 4) in children with bronchial asthma. Methods Forty asthmatic children and 10 normal children with obvious family history and allergy were enrolled in this study. PCR amplification and single strand conformation polymorphism (SSCP) amplification were used to screen the IL 4 proximal promoter fragment. The recombinant plasmid pIL 4 Jx2 of normal control and abnormal PCR products was further constructed, and the recombinant plasmid was sequenced by dideoxy chain termination method. Results In the SSCP analysis of 40 children, seven abnormal bands were found. DNA sequencing showed that four of the mutation sites were located within or adjacent to known regulatory elements: one patient, -29 C, was replaced by A, and the mutation was located just above IL 4 positive regulatory element I (PRE I ); Two patient negative regulatory element Ⅱ (NRE Ⅱ) elements adjacent to C replaced by T, TATA front increased by a G; STATT elements in a patient missing five ATTTT nucleotide. Conclusion The DNA sequence of IL 4 promoter in children with allergic asthma is polymorphic, which may be related to the abnormal expression of IL 4 gene and the pathogenesis of asthma.