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目的 为进一步探索酪氨酸酶相关蛋白 -1(TRP 1)人源B细胞表位 ,利用甲醇营养型酵母Pichiapas toris表达系统表达TRP 1的B细胞表位区。方法 将本实验室已构建好的质粒 pUC19/TRP 1进行双酶切 ,将目的片段亚克隆至带有 6His标签的 pRSETA载体 ,将 6His融合的TRP 1整体PCR扩增出来 ,克隆到酵母表达载体pPIC3 .5K上 ,构建成重组质粒 pPIC3 .5K/6His TRP1。该质粒转化酵母菌GS115 ,经G418筛选得到高拷贝转化子。转化菌体经Mut表型鉴定后 ,用含 0 .5 %甲醇的培养基诱导表达 ,表达产物利用Ni NTAagarose柱通过金属螯合亲和层析进行纯化后 ,测定其生物学活性。结果 通过 4天的诱导 ,该系统成功表达了 6His TRP1蛋白 ,经亲和层析纯化后扫描分析重组蛋白分子量约为 18kD ,纯度可达 96%。Westernblotting及ELISA实验证实 ,表达产物具有良好的抗原性和特异性。生物学活性检测证实其具有结合白癜风病人IgG的能力。 结论 在Pichiapastoris表达系统中 ,获得了具有生物学活性的可溶性重组TRP 1的B细胞表位肽段 ,为深入研究TRP -1人源表位及白癜风的发病机制、免疫治疗及恶性黑素瘤的免疫治疗奠定了基础。
Objective To further explore the human B cell epitope of tyrosinase related protein - 1 (TRP 1), the B cell epitope region of TRP 1 was expressed by using the methanol - nutrient yeast Pichiapas toris expression system. Methods The constructed plasmid pUC19 / TRP 1 in our laboratory was double-digested. The target fragment was subcloned into pRSETA vector carrying 6His tag. The 6His-fused TRP 1 was amplified by PCR and cloned into yeast expression vector pPIC3 .5K, constructed into a recombinant plasmid pPIC3 .5K / 6His TRP1. The plasmid was transformed into yeast GS115 and screened by G418 to obtain high copy transformants. The transformed cells were identified by the Mut phenotype and expressed in medium containing 0.5% methanol. The expressed product was purified by metal chelate affinity chromatography using Ni NTA agarose column and its biological activity was determined. Results After 4 days of induction, the system successfully expressed 6His TRP1 protein. The molecular weight of recombinant protein was about 18kD after purification by affinity chromatography and the purity was up to 96%. Westernblotting and ELISA experiments confirmed that the expression product has good antigenicity and specificity. Biological activity tests confirmed its ability to bind IgG patients with vitiligo. Conclusions In the Pichia pastoris expression system, a B cell epitope peptide of soluble recombinant TRP 1 with biological activity was obtained. In order to further study the pathogenesis of TRP -1 human epitope and vitiligo, immunotherapy and malignant melanoma Immunotherapy laid the foundation.