蜜蜂主要变应原酸性磷酸酯酶基因的克隆及原核表达载体的构建

Molecule cloning of a major allergen acid phosphatase(Apim3) gene from honeybees and construction of a prokaryotic expression vector

目的克隆蜜蜂Apis cerana cerana主要变应原酸性磷酸酯酶(Api m3)基因,构建其原核表达载体。方法根据已知序列设计带有酶切位点的特异性引物,以提取的蜜蜂总RNA为模板RT-PCR扩增目的基因,并进行序列分析。将基因片段亚克隆到PET-28a表达载体上。结果获得蜜蜂Api m3基因,构建了其原核表达载体。基因分析显示该基因全长1 167 bp,编码388个氨基酸,推测分子质量单位为45.279 9 ku,等电点为5.53。序列分析显示与Gen-Bank中已知基因的同源性为97%。结论成功克隆蜜蜂Api m3基因并构建原核表达载体,对进一步进行变应原表达和免疫学活性鉴定有积极意义。

Objective To clone a major allergen acid phosphatase（Apim3） gene from honeybees（Apis cerana cerana） and construct a prokaryotic expression vector.Methods Special primers were designed on the basis of the reported gene.Total RNA was extracted from bees.RT-PCR was used to amplify the Apim3 gene and sequence analysis was performed.Fragments were subcloned into the expression vector pET-28a.Results The A pim3 gene was obtained from honeybees and a prokaryotic expression vector was constructed.Analysis revealed that the gene consisted of 1 167 nucleotides encoding 388 amino acids with a molecular weight of 45 279.9 and pI of 5.53.The gene had homology of 97% with the gene reported in GenBank.Conclusion The honeybee Apim3 gene was cloned and a prokaryotic expression vector was successfully constructed.This should prove to be of considerable significance in determining the expression of allergens and immunological activity.