采用简并引物克隆葎草花粉过敏原全长同源cDNA  

Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens (Lour.) Merr by degenerate primer

作　　者：陶爱林[1] 何韶衡[1] 张利达[2] 陈章权[1] 李东栋[1] 

机构地区：[1]汕头大学医学院变态反应与炎症研究所,广东汕头515031 [2]上海交通大学复旦-交大-诺丁汉植物生物技术研究中心,上海200030

出　　处：《细胞与分子免疫学杂志》2004年第1期99-103,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：广东省自然科学基金课题资助 (No.0 3461 7)

摘　　要：目的 :建立一套稳定可靠的方法 ,对过敏原性物种中的过敏原同源基因进行快速克隆。方法 :在分析生物信息数据库中积累的大量过敏原序列同源性的基础上 ,设计简并引物 ,基于高质量草花粉RNA ,逆转录合成cDNA。采用Touch down方式在cDNA池中进行选择性PCR扩增。同时借助梯度PCR程序 ,对引物扩增的简并性作进一步强化 ,并结合RACE技术获取全长cDNA ,进而对草花粉中的过敏原同源基因进行克隆。结果 :成功地获得 3个全长cDNA克隆。序列分析显示 ,这些基因与已知过敏原的基因序列相似性高达79%～ 85 % ,初步认定其为泛过敏原肌球蛋白抑制蛋白 (pro filin)的同源基因。对比RACE技术获得的相应基因的全长序列发现 ,这些序列在引物结合处与简并引物序列之间存在 4个碱基的差异 ,提示采用Touchdown方式的梯度PCR程序 ,可使引物的简并性得到进一步扩展。结论 :简并引物与Touch down梯度PCR相结合的方法 。AIM: To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants. METHODS: Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences. RESULTS: Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%～85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR. CONCLUSION: Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.

关 键 词：简并引物 梯度PCR 基因克隆 过敏原 同源基因 

分 类 号：R282.71[医药卫生—中药学]