CAJ | 학술논문

目的：克隆表达反枝苋花粉中泛变应原肌动蛋白抑制蛋白（profilin），并鉴定其免疫学活性。方法利用RT-PCR 结合 RACE 技术克隆反枝苋花粉泛变应原 profilin 的全长基因，并进行序列分析。设计带有酶切位点的特异性引物，采用 RT-PCR 获得整个反枝苋花粉 profilin 的开放阅读框，将其与 pET28a 载体连接并转化大肠杆菌BL21（DE3）进行诱导表达，Ni2＋亲和层析柱纯化重组蛋白，采用 Western-blot 检测其 IgE 结合活性。结果cDNA核苷酸测序表明反枝苋花粉 profilin 的全长基因由675个碱基组成，开放阅读框为399 bp，编码131个氨基酸。重组反枝苋花粉 profilin 在大肠杆菌中可溶性表达，进一步经 Western-blot 检测具有良好的 IgE 结合活性。结论成功地克隆和表达了反枝苋花粉 profilin，并具有良好的 IgE 应答免疫活性。
목적：극륭표체반지현화분중범변응원기동단백억제단백（profilin），병감정기면역학활성。방법이용RT-PCR 결합 RACE 기술극륭반지현화분범변응원 profilin 적전장기인，병진행서렬분석。설계대유매절위점적특이성인물，채용 RT-PCR 획득정개반지현화분 profilin 적개방열독광，장기여 pET28a 재체련접병전화대장간균BL21（DE3）진행유도표체，Ni2＋친화층석주순화중조단백，채용 Western-blot 검측기 IgE 결합활성。결과cDNA핵감산측서표명반지현화분 profilin 적전장기인유675개감기조성，개방열독광위399 bp，편마131개안기산。중조반지현화분 profilin 재대장간균중가용성표체，진일보경 Western-blot 검측구유량호적 IgE 결합활성。결론성공지극륭화표체료반지현화분 profilin，병구유량호적 IgE 응답면역활성。
Objective To clone and express Amaranthus retroflexus pollen profilin,and to eval-uate the immunologic activity of the allergen.Methods The full-length profilin gene from Ama-ranthus retroflexus pollen was cloned using RT-PCR and RACE.Then the sequence was ana-lyzed.The open reading frame (ORF)of panallergen profilin was amplified by RT-PCR using spe-cific primers with restriction sites.The profilin gene was cloned into pET28a vector,expressed in E.coli BL21(DE3),and purified by Ni2+ affinity chromatography.The IgE-binding capacity of the recombinant protein was analyzed by Western-blot.Results Nucleotide sequencing of the cDNA revealed that the cloned fragment of Amaranthus retroflexus pollen profilin was 675 bp in length with a 399 bp ORF encoding 131 amino acid residues.The recombinant protein was expressed in E.coli as a soluble protein which showed a strong IgE reactivity.Conclusion The profilin from the Amaranthus retroflexus pollen was successfully cloned and expressed with a strong IgE reac-tivity.