支气管哮喘患者白细胞介素-4近侧启动子区克隆和序列分析

目的 探讨哮喘患者白细胞介素-4（IL-4）过度表达的可能调控机制。方法 选取4例有明显家族史的过敏性哮喘患儿和1例正常儿童，采用聚合酶链反应（PCR）扩增IL-4近侧启动子片段，PCR产物经XbaI、HindⅢ限制性内切酶进行双酶切后，回收酶切片段，再与经过相同双酶切的载体Jx2相连接，制备重组质粒，然后经转化、筛选、酶切鉴定得到重组质粒pIL-4-Jx2，最后采用双脱氧终止法对重组质粒进行序列测定。结果 （1）正常儿童IL-4近侧启动子序列与基因库序列相同。（2）1例患儿-229位C被A所替代。该变异恰好位于正性调节元件-I之内，结论 过敏性哮喘患者IL-4近侧启动子区DNA片段被成功克隆，并发现1例患者IL-4调控元件内一未曾报道过的变异位点，这有助于对哮喘者IL-4基因异常表达调控机制的研究。

Cloning and sequencing of IL-4 proximal promoter of asthmatic patients

Objective To investigate the possible regulatory mechanism ofIL-4 overexpression in asthmatic patients.Methods The IL-4 proximal promoter segments were obtained by polymerase chain reaction (PCR) with genomic DNA from 4 patients with dominantly allergic familial history and a healthy child as template. The PCR segments were digested with Xba Ⅰ and Hind Ⅲ and then recovered from agarose gels. The recovered segments were ligated with vector Jx2 that had been digested previously using the same restriction enzyme to prepare recombinant plasmids. The recombinant plasmids pIL-4-Jx2 were obtained after transformation, selection and restriction enzyme analysis. The PCR inserts were sequenced by dideoxy chain termination method in the end. Results The IL-4 proximal promoter sequences of the normal child were the same as the sequences of the gene pool. A C to A transversion located at -229 position was just within PRE-I element in one patient.Conclusion The IL-4 proximal promoter segments of asthmatic patients were successfully cloned and sequenced. A new mutation within PRE-I element was found. It was useful to detect the mechanism of the aberrant expression of IL-4 in asthmatic patients.