尘螨变应原Der p2基因克隆及原核表达 |
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引用本文: | 刘良,周鹰,彭江龙,崔玉宝. 尘螨变应原Der p2基因克隆及原核表达[J]. 中国媒介生物学及控制杂志, 2008, 19(4): 320-323 |
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作者姓名: | 刘良 周鹰 彭江龙 崔玉宝 |
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作者单位: | 1. 盐城卫生职业技术学院卫生技术研究所,盐城,224006 2. 海南医学院寄生虫学教研室 3. 盐城卫生职业技术学院卫生技术研究所,盐城224006;海南医学院寄生虫学教研室 |
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基金项目: | 国家自然科学基金 , 海南省卫生厅科研课题 |
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摘 要: | 目的 原核表达尘螨主要变应原DerP 2。方法分离屋尘螨总RNA,根据GenBank已公布的DerP2核酸序列设计引物用RT-PcR扩增DerP2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 coliBL21(DE3)并用IFFG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DerP2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DerP2原核表达获得成功,为进一步生产重组变应原奠定了基础。
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关 键 词: | 尘螨 DerP2 克隆 原核表达 |
Cloning and expression of Der p 2 allergen of Dermatophagoides pteronyssinus |
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Affiliation: | LIU Liang , ZHOU Ying , PENG Jiang- long, CUI Yu-bao. (Yancheng College of Medical Occupation and Technology, Yancheng 224006, China) |
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Abstract: | 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. |
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Keywords: | House dust mite Der p 2 Clone Prokaryotic expression |
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