从葎草花粉cDNA表达文库中筛选过敏性哮喘特异性变应原

目的 从葎草花粉cDNA表达文库中筛选过敏性哮喘特异性变应原.方法 应用葎草花粉过敏性哮喘患者血清对自行构建的葎草花粉cDNA λTripIEx2噬菌体表达文库进行免疫学筛选,提取阳性噬菌体DNA,EeoR I和HindⅢ双酶切、琼脂糖凝胶电泳鉴定后进行DNA序列测定、重复序列分析和同源性分析.结果 经过3轮筛选,从cDNA表达文库中筛选获得3个葎草花粉特异性变应原cDNA克隆,其序列大小分别为868、550和592 bp.测序结果表明这3个克隆代表3个不同的cDNA序列,重复序列分析未发现重复序列,同源性分析表明与现有的基因均无高度同源性.结论 所获得的与过敏性哮喘相关的3个葎草花粉特异性变应原cDNA克隆可能为新基因,此发现为进一步制备重组变应原疫苗或核酸疫苗打下了良好基础.

Screening specific allergens of allergic asthma from DNA expression library of humulus pollen

Objective To screen the allergic asthma-specific allergen from complementary DNA (cDNA)expression library of humulus pollen.Methods Screened the humulus pollen λTripIEx2 cDNA expression library which had been constructed using the method of immunological screening with patients' blood serum of humulus pollen allergic asthma,extracted the positive phage DNA and the positive phage DNA.and identitied by double restriction enzyme with EcoR Ⅰ,Hind Ⅲ and agarose gel electrophoresis.Then the DNA fragments were sequenced and the repeats and sequence homology were analyzed.Results After 3 rounds of screening,three specific allergic cDNA clone of humulus pollen were obtained and the sequences were 868,550 and 592 base pairs respectively.The results of sequencing showed that these three clones represented different cDNA sequences.The repeats analysis found no repeats and these clones had no high homology with any known gene.Conclusions Three specific allergic cDNA clones of humulus pollen obtained may be new genes.It provides a rational basis for constructing a recombinant allergen or nuclear acid vaccine.