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目的克隆表达粉尘螨第五组变应原(Dermatophagoides farinae,Derf5)基因,并鉴定纯化蛋白免疫原性。方法提取活粉尘螨总RNA,扩增Derf5片段,PCR产物与克隆载体pMD18-T连接,转化入大肠埃希菌JM109,经酶切及测序鉴定获得pMD18-Der f5阳性菌株,再提取质粒进行双酶切,与表达载体pET-30a(+)连接,转化入大肠埃希菌BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)鉴定其表达效果,Ni-IDA亲和层析柱纯化蛋白,利用尘螨病人血清鉴定其免疫原性。结果构建了重组质粒pMD18-Derf5和pET30a-Der f5,SDS-PAGE结果表明Der f5基因在BL21中获得良好的可溶性表达,蛋白质分子量与理论值相符,纯化的蛋白与病人血清有良好的IgE结合活性。结论获得广州地区Der f5的原核表达载体,高效表达纯化重组蛋白,初步鉴定了该蛋白的免疫原性。
Objective To clone and express Dermatophagoides farinae (Derf5) gene and identify the immunogenicity of the purified protein. Methods Total RNA was extracted from Dermatophagoides pteronyssinus and Derf5 was amplified. The PCR product was cloned into pMD18-T and transformed into Escherichia coli JM109. The pMD18-Der f5 positive strain was identified by restriction enzyme digestion and sequencing. Then ligated with the expression vector pET-30a (+) and transformed into E. coli BL21 for expression under induction of IPTG. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (Western blotting) to identify its expression, Ni-IDA affinity chromatography purification of protein, the use of dust mite patient serum to identify its immunogenicity. Results The recombinant plasmids pMD18-Derf5 and pET30a-Der f5 were constructed. The results of SDS-PAGE showed that the Der f5 gene was successfully expressed in BL21. The protein molecular weight was consistent with the theoretical value. The purified protein had good IgE-binding activity . Conclusion The prokaryotic expression vector Der f5 in Guangzhou was obtained and the recombinant protein was highly expressed. The immunogenicity of the protein was preliminarily identified.