粉尘螨过敏原Der f 13基因克隆表达与免疫原性鉴定

Gene Cloning,Expression and Immunogenicity Characterization of Allergen Der f 13 from Dermatophagoides Farinae

目的克隆表达粉尘螨过敏原Der f 13,并鉴定其免疫原性。方法提取粉尘螨总RNA,通过RT-PCR获得Der f 13的cDNA片段。根据已知Der f 13序列设计出表达引物,再通过已获得的cDNA和引物进行PCR扩增,将扩增产物连接到T载体上并转入克隆菌Top 10中,涂板、培养,挑选阳性菌落,LB/AMP培养送测序。将测序正确的基因转入表达载体PET32中,经酶切鉴定测序再转入表达菌BL21中,表达Derf 13蛋白,纯化、鉴定所得蛋白,并通过Western-blot等方法检测其免疫学活性。结果 RT-PCR结果显示,Der f 13基因片段大小为410 bp;同源性分析表明:本实验所克隆的Der f 13基因与NCBI数据库的Der f 13基因的同源性为95%;SDS-PAGE结果显示,重组质粒PET32-Der f 13在BL21(DE3)中高效表达,重组蛋白分子质量为35 ku,表达后的重组蛋白主要以可溶性蛋白形式存在,经与尘螨过敏患者血清反应,检测出重组蛋白有较强免疫学活性。结论克隆出Der f 13基因,表达和纯化出目的蛋白,并具有免疫原性,为过敏性疾病的诊断和免疫治疗奠定理论基础。

Objective To clone and express the antigen Der f 13 from the dust mite Dermatoph-agoides farinae,and to identify its immunogenicity.Methods The total RNA was extracted from dust mites and Der f 13 gene fragments were amplified by RT-PCR.The cDNA synthesis reaction was used for PCR amplification with specific primers.The amplified products were inserted into T vector,and then were transformed into the cloning bacteria Top 10.The positive colonies were se-lected by LB/AMP culture medium and the sequence was detected.The Der f 13 gene sequences confirmed by DNA sequencing were connected to pET32a.The recombinant plasmids were trans-fected into E.coli strain BL21 to express Der f 13 protein.The recombinant protein was purified by affinity chromatography and the allergenicity was assayed by Western-blot.Results RT-PCR analysis showed that the size of Der f 13 gene fragment was 400 bp approximately.Homology a-nalysis showed that the gene sequence of Der f 13 cloned in this study shared 95% homology with that registered in NCBI.SDS-PAGE analysis showed that the pET32a-Der f 13 was highly ex-pressed in BL21.The recombined protein was soluble with a relative molecular weight of 30 kD. In addition,the protein showed a high ability to bind serum IgE from dust mite-allergic patients, suggesting that the recombined Der f 13 had a high immunogenicity.Conclusion The Der f 13 gene has been cloned,expressed and purified and the recombinant Der f 13 protein shows appro-priate immunogenicity.The study lays a theoretical foundation for the diagnosis and treatment of allergic disease.