哮喘患儿IL-4近侧启动子区克隆和多态性分析

Cloning and polymorphism analysis of IL-4 proximal promoter of asthmatic patients

目的 对哮喘症患儿白细胞介素 4 (IL 4 )近侧启动子区进行克隆并分析其DNA序列的多态性。方法 对 4 0名有明显家族及过敏史的哮喘患儿和 10名正常儿童 ,以多聚酶链式反应(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 在对 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 4处变异位点位于已知的调控元件之内或毗邻位点 ,1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 )之内 ;2名病人负性调节元件 Ⅱ (NRE Ⅱ )毗邻C被T所替代 ,TATA框前增加 1个G ;1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

Objective To clone and study the polymorphism of interleukin 4 (IL 4) proximal promoter from asthmatic patients. Methods The IL 4 proximal promoter segments were amplified and selected by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) with genomic DNA from ten healthy children and forty patients with dominantly allergic familial history were used as template. The selected PCR segments were cloned into recombinant plasmids pIL 4 Jx2. The PCR inserts were sequenced by dideoxy chain termination method in the end. Results Seven aberrant bands were identified in SSCP analysis from forty asthmatic patients. The sequencing results showed that four variant sites were found within or adjacent to the known IL 4 regulatory element. C→A transversion located at 229 position was just within positive regulatory element Ⅰ (PRE Ⅰ) in one patient. C→T transition adjacent to negative regulatory element Ⅱ ( NRE Ⅱ ) and an extra G adjacent to TATA box were found in two patients. A five bases nucleotide deletion was found near signal transducers and activators of transcription 6 responsive element(STAT 6 RE) in one patient. Conclusion There were polymorphisms of IL 4 proximal promoter from allergic asthmatic patients and these polymorphisms might result in aberrant expression of IL 4 gene and asthma. [