粉尘螨过敏原Derf13基因克隆表达与免疫原性鉴定

粉尘螨过敏原Derf13基因克隆表达与免疫原性鉴定
王媛媛;林建立;黄娜娜;巴金戈;刘志刚;刘晓宇
【期刊名称】《南昌大学学报（医学版）》
【年(卷),期】2016(000)001
【摘要】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。

%ABSTRACT: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.
【总页数】5页(P21-25)
【作者】王媛媛;林建立;黄娜娜;巴金戈;刘志刚;刘晓宇
【作者单位】深圳大学医学院过敏反应与免疫学研究所，广东深圳 518060;深圳
大学医学院过敏反应与免疫学研究所，广东深圳 518060;深圳大学医学院过敏反
应与免疫学研究所，广东深圳518060;深圳大学医学院过敏反应与免疫学研究所，
广东深圳 518060;深圳大学医学院过敏反应与免疫学研究所，广东深圳 518060;深圳大学医学院过敏反应与免疫学研究所，广东深圳 518060
【正文语种】中文
【中图分类】R392.8
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