摘要
目的克隆并表达短穗鱼尾葵花粉中泛变应原肌动蛋白抑制蛋白(profilin)。方法利用RT-PCR结合RACE技术克隆短穗鱼尾葵花粉中泛变应原profilin的全长基因,并进行序列分析。然后设计带有酶切位点的特异性引物,采用RT-PCR获得整个短穗鱼尾葵花粉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,通过Ni2+亲和层析柱对重组蛋白进行纯化,采用Western-blot检测其IgE结合活性。结果克隆获得了短穗鱼尾葵花粉profilin的全长基因,由608个碱基组成,开放阅读框为396个碱基(包括终止密码子),编码131个氨基酸。该序列编码的蛋白为小分子量酸性蛋白,等电点为4.52,相对分子质量约为14200。此序列已被GenBank收录,登陆号为EF173600。重组短穗鱼尾葵花粉profilin在大肠杆菌中高效的表达,进一步经Ni2+亲和层析柱纯化后经Western-blot检测具有良好的免疫学活性。结论成功地克隆和表达了短穗鱼尾葵花粉profilin,为短穗鱼尾葵花粉过敏的诊断和免疫治疗奠定了基础。
Objective To clone, express and characterize the panallergen profilin from the pollen of Caryota mitis Lour. Methods RT-PCR and RACE methods were applied to clone the full-length panallergen gene from Caryota mitis pollen. The sequence of the insert was analyzed. The ORF of profilin of Caryota mitis pollen was amplified by RTPCR using specific primers and cloned into the expression vector pET-28a. The recombinant Caryota mitis pollen profilin was expressed in Escherichia coli BI221 （DE3）. The recombinant protein was purified by affinity chromatography using Ni^2＋ coupled sepharose. IgE reactivity of recombinant Caryota mitis pollen profilin was investigated by western blot. Results The Caryota mitis pollen profilin gene was cloned. The cloned fragment was 608 bp in length, with an 396 bp open reading frame coding for 131 amino acids. Sequence analysis showed that the encoded protein was a acidic protein with an estimated molecular mass of 14.2 kDa and a pI of 4.52. The GenBank accession number of this clones was EF173600. The recombinant protein was expressed in Escherichia coli BL21 （DE3） and purified by Ni^2＋ -sepharose column. Immunoassay showed that the recombinant allergen has good IgE binding capacity. Conclusion The profilin of Caryota mitis pollen was cloned and expressed successfully in E. coli BL21 （DE3）, which will be used for further study on Caryota mitis pollen related allergy.
出处
《热带医学杂志》
CAS
2007年第7期618-621,632,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(No.30070702)
广东省自然科学基金(No.020738)
广东省科技重点专项(No.2003A3080502)
深圳市科技计划项目(No.200326)。
