尘螨变应原Der p 2基因克隆及原核表达

Cloning and expression of Der p 2 allergen of Dermatophagoides pteronyssinus

目的原核表达尘螨主要变应原Der p 2。方法分离屋尘螨总RNA,根据GenBank已公布的Der p 2核酸序列设计引物用RT-PCR扩增Der p 2编码基因,克隆至pMD19-T载体、亚克隆至表达载体pET28a(+),将表达质粒转化至Escherichia coli BL21(DE3)并用IPTG诱导表达,并对表达产物进行免疫印迹鉴定。结果成功构建了表达质粒pET28a(+)-Derp2,SDS-PAGE和Western blotting显示原核表达获得成功。序列分析表明所获得的Der p 2编码基因与参考序列同源性达99.54%,推测其编码氨基酸130个,相对分子质量约为14348.5。结论尘螨变应原Der p 2原核表达获得成功,为进一步生产重组变应原奠定了基础。

Objective To clone, express and characterize Dermatophagoides pteranyssinus major allergens. Methods Based on Der p 2 nucleotide sequence published in the GenBank, we designed primers and amplified the cDNA fragment coding Der p 2 allergen from D. pteronyssinus by RT-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a（ ＋ ）, expressed in Escherichia coli BL21 and identified by Western blotting. Results The cDNA coding Derp 2 allergen of adult D. pteronyssinus was cloned, sequenced and expressed successfully. Sequence analysis showed the gene homology with Der p 2 reported in GenBank was 99.54 %, which probably encode a protein with 130 amino acids and its molecular weight was 14 348.5. Conclusion The cDNA coding Derp 2 allergen of D. pteronyssinus were cloned and expressed successfully, which provided a foundation for the production of recombined allergenst.