曼氏血吸虫虫卵蛋白IPSE基因合成、表达及特性分析

目的合成并表达曼氏血吸虫虫卵白介素4诱导物(IPSE)基因,并对其免疫特性进行分析。方法应用重叠PCR方法合成曼氏血吸虫IPSE基因,将其定向克隆至表达载体pET32a(+)的硫氧还蛋白(Trx)序列下游,构建重组表达质粒IPSE/pET32a(+)。将重组质粒转化至大肠埃希菌BL21(DE3)中,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,表达Trx-IPSE融合蛋白。大量制备表达产物,用镍螯合亲和层析胶在变性条件下纯化重组Trx-IPSE融合蛋白。用大量PBS对变性融合蛋白进行透析,获得部分复性的可溶性Trx-IPSE融合蛋白。应用蛋白质印迹(Westernblotting)分析其是否能被慢性日本血吸虫病患者血清中IgG抗体所识别,及其与健康人血清中IgE抗体的结合能力。结果人工合成的IPSE基因序列与天然基因序列完全一致。经IPTG诱导,SDS-PAGE分析,含重组质粒的转化子细菌能表达相对分子质量(Mr)约35700的蛋白分子,与预计Trx-IPSE融合蛋白的相对分子质量大小相符。Westernblotting分析结果显示,变性的和复性的Trx-IPSE融合蛋白均能被慢性日本血吸虫病患者血清中IgG抗体识别,融合蛋白Trx-IPSE能与健康人血清中IgE抗体非特异性结合。结论曼氏血吸虫IPSE基因合成获得成功,重组表达的IPSE具有与天然抗日本血吸虫抗体反应和与IgE非特异性结合的能力。

Gene Synthesis,Expression and Characterization of Egg Protein IPSE of Schistosoma mansoni

Objective To synthesize and express the gene of egg protein IPSE (IL-4-inducing principle of Schistosoma mansoni eggs) and evaluate its immunologic characteristics. Methods The IPSE gene of S. mansoni was synthesized by overlapping PCR, and confirmed by DNA sequencing. The recombinant plasmid IPSE-pET32a(+) was cons- tructed by inserting the gene of IPSE into expression vector pET32a(+) at the downstream of thioredoxin(Trx) coding sequence. The recombinant plasmid IPSE-pET32a(+) was transformed into E. coli...