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目的构建Der f1的T细胞表位融合肽基因的原核表达载体并表达鉴定。方法利用基因工程方法构建Der f1的T细胞表位融合肽嵌合基因,命名为Der f1T,并插入到原核表达载体p ET28a(+)中,形成重组原核表达载体p ET28a(+)-Der f1T,进行双酶切和测序验证后的阳性克隆转化到E.coli BL21(DE3)诱导其表达。制样进行SDS-PAGE分析表达产物,再进行纯化,并Western bloting鉴定表达的蛋白。ELISA法测定重组蛋白对粉尘螨过敏的患者血清Ig E抗体的结合能力。结果双酶切和测序结果说明原核表达载体p ET28a(+)-Der f1T构建成功;SDS-PAGE说明Der f1T诱导且纯化成功;Western bloting进一步说明Der f1T蛋白纯化成功;ELISA试验结果说明Der f1T对粉尘螨过敏的患者血清中Ig E结合能力与Der f1相比明显下降(P<0.05)。结论成功表达Der f1的T细胞表位重组蛋白,为后续粉尘螨过敏患者提供特异性免疫治疗奠定基础。
Objective To construct prokaryotic expression vector of Der f1 T cell epitope fusion peptide gene and express it. Methods The gene encoding Der f1 T cell epitope fusion gene was constructed by gene engineering and named as Der f1T, which was inserted into the prokaryotic expression vector p ET28a (+) to form the recombinant prokaryotic expression vector p ET28a (+) - Der f1T After double digestion and sequencing, the positive clones were transformed into E.coli BL21 (DE3) to induce their expression. Samples were analyzed by SDS-PAGE analysis of expression products, and then purified, and Western bloting identified the expression of the protein. ELISA method for the determination of recombinant proteins on dust mite allergy patients serum Ig E antibody binding capacity. Results The results of double enzyme digestion and sequencing showed that the recombinant plasmid p ET28a (+) - Der f1T was constructed successfully. SDS-PAGE showed that Der f1T was induced and purified successfully. Western bloting further demonstrated that Der f1T protein was purified successfully. Serum Ig E binding capacity of patients with dust mite allergy was significantly lower than that of Der f1 (P <0.05). Conclusion The successful expression of Der f1 T-cell epitope recombinant protein provides the basis for the follow-up immunodeficiency virus-specific immunotherapy.