TRP-1 B细胞表位区在酵母中的表达纯化及生物学活性研究

Expression Purification and Biological Activity Analysis of B Cell Epitope Domain of Human Tyrosinase Related Protein-1(TRP-1) in Pichia Pastoris

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 study the expression of human tyrosinase related protein-1(TRP-1) in Pichia pastoris and to study the human B cell epitope of tyrosinase related protein-1(TRP-1).Methods Subcloning the gene of TRP-1 into vector pRSETA with the tag of 6 His from the plasmids pUC19/TRP-1,amplifying 6His TRP1 by PCR,after confirmed by DNA sequence analysis,the gene was inserted into the Pichia pastoris expression vector pPIC3.5K containing AOX1 promoter,the recombinant plasmids pPIC3.5K/6His-TRP1 was constructed and transformed into GS115.After in vivo screening of mutiple inserts by G418 and screening for Mut+ transformants,the Mut+ mutiple inserts were induced by 5 ml/L methanol.Results After 4 days of methanol induction,the expressed 6His-TRP1 fusion proteins were analyzed by Coomassie-stained SDS-PAGE.Western blotting ELISA assay proved the expressed 6His TRP-1 having good antigenicity and high specificity and having good biological activity to bind IgG of vitiligo patients.The recombinant protein was purified with Ni-NTA agarose affinity chromatography.the molcelluer weight is about 18KD and the purity is about 96%.Conclusion Soluble B cell epitope peptide of TRP-1 with good biological activity were successfully achieved in Pichia pastoris expression system,which will be not only the base of deeply studying the human B cells epitope of tyrosinase related protein-1(TRP-1) and searching for the etiology of vitiligo,but also the base of immune therpy of vitiligo and malignant melanoma.