曼氏血吸虫虫卵蛋白IPSE基因合成、表达及特性分析  

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

作　　者：谢曙英[1,2] 陈红根 余传信[2] 殷旭仁[2] 华万全[2] 曾小军[2] 梁幼生[2] 高琪[2] 

机构地区：[1]江西省寄生虫病防治研究所,南昌330046 [2]江苏省寄生虫病防治研究所,卫生部寄生虫病预防与控制技术重点实验室,无锡214064

出　　处：《中国寄生虫学与寄生虫病杂志》2010年第2期89-93,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30671833;30471515);国家"十一五"科技重大专项(No.2008ZX10004-011);江西省卫生厅科技计划(No.20092011)~~

摘　　要：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。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 BL21（DE3） and followed by expression of the protein induced by IPTG. Large-scale fusion protein was prepared and purified with Ni affinity chromatography gel under denaturing conditions. A small amount of soluble Trx-IPSE was obtained by dialyzing the fusion protein in a large volume of PBS. Western blotting was used to detect if the recombinant IPSE was recognized by the IgG antibody in the pooled patient sera of schistosomiasis japonica and its binding capacity to the non-specific IgE antibody in the sera of healthy persons. Results DNA sequencing confirmed that the nucleotide sequence of synthesized IPSE gene was completely identical to the native one. SDS-PAGE showed that the recombinant plasmid IPSE/pET32a（＋） expressed a fusion protein with an Mr 35 700 after being induced by IPTG. The pure fusion protein Trx-IPSE reacted positively with the pooled sera of schistosomiasis patients under either denaturing or renaturing conditions. The protein Trx-IPSE also reacted with the non- specific IgE in the sera of healthy persons. Conclusion The IPSE gene of Schistosoma mansoni has been synthesized,and the recombinant can react with natural antibody IgG against S. japonicum and non-specifically bind to IgE antibody.

关 键 词：曼氏血吸虫 虫卵蛋白 血吸虫虫卵白介素4诱导物 基因合成 功能分析 

分 类 号：R383.24[医药卫生—医学寄生虫学]