豚草花粉泛过敏原同源基因克隆与序列分析

目的　克隆豚草花粉泛过敏原同源基因。方法　采用生物信息学分析方法对众多的花粉过敏原基因进行序列同源性比较 ,以序列保守区域为依据设计合成简并引物 ,在特殊的RT PCR条件下 ,结合RACE技术对豚草花粉中的过敏原全长基因进行克隆 ;通过Northern杂交及序列分析初步确定基因产物是否为花粉过敏原。结果　获得了 3个新的全长基因。序列分析显示 :所得过敏原同源基因与数十种不同种属来源的过敏原肌动蛋白结合蛋白 (profilin)具有较高的同源性 ,初步认定其为泛过敏原 ,并暂命名为Amba 8(t)。Northern杂交证实该基因在花粉中表达。结论　采用本研究方法成功地在豚草花粉中克隆到 3个泛过敏原基因 ,为豚草花粉重组过敏原的蛋白质表达及标准化奠定了物质基础

Cloning and sequencing of homologous panallergen genes in pollen of short ragweed (Ambrosia artemisiifolia L.)

Objective To clone homologous panallergen genes in the pollen of short ragweed ( Ambrosia artemisiifolia L.). Methods Bioinformatic method was used for the comparative analysis of numerous homologous pollen allergen gene sequences. Conservative domains among the sequences were determined for degenerate primer designing. The defined PCR conditions and RACE (rapid amplification of cDNA ends) method were adopted to clone the full length allergen genes from short ragweed pollen. Northern blot and multiple alignments were used to identify the properties of the gene product. Results Three novel cDNA clones were obtained, and their pollen sources were confirmed by Northern blot. Sequence analysis showed that all three genes cloned shared a high homology with the panallergen profilins from a number of different genera. The deduced proteins were therefore regarded as panallergen and named as Amb a 8(t). Conclusion We cloned three novel panallergen genes from short ragweed pollen by adopting homology sequence method, which will be used as a key material for the expression of allergen proteins applied in diagnosis and treatment of pollen allergic patients.