Xie Shu-ying, Chen Hong-gen, Yu Chuan-xin, Yin Xu-ren, Hua Wan-quan, Zeng Xiao-jun, Liang You-sheng, Gao Qi
Jiangxi Provincial Institute of Parasitic Diseases, Nanchang 330046, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Apr;28(2):89-93.
To synthesize and express the gene of egg protein IPSE (IL-4-inducing principle of Schistosoma mansoni eggs) and evaluate its immunologic characteristics.
The IPSE gene of S. mansoni was synthesized by overlapping PCR, and confirmed by DNA sequencing, The recombinant plasmid IPSE-pET32a(+) was constructed 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.
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 35700 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 nonspecific IgE in the sera of healthy persons.
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.
合成并表达曼氏血吸虫卵蛋白IPSE（曼氏血吸虫卵白细胞介素4诱导因子）基因，评估其免疫学特性。
采用重叠PCR法合成曼氏血吸虫IPSE基因，并进行DNA测序验证。将IPSE基因插入表达载体pET32a(+)中硫氧还蛋白（Trx）编码序列下游，构建重组质粒IPSE-pET32a(+)。将重组质粒IPSE-pET32a(+)转化至大肠杆菌BL21(DE3)，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达蛋白。在变性条件下，用镍亲和层析凝胶制备并纯化大量融合蛋白。通过将融合蛋白在大量磷酸盐缓冲液（PBS）中透析获得少量可溶性Trx-IPSE。采用蛋白质免疫印迹法检测重组IPSE是否能被日本血吸虫病患者混合血清中的IgG抗体识别及其与健康人血清中非特异性IgE抗体的结合能力。
DNA测序证实合成的IPSE基因核苷酸序列与天然序列完全一致。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示，重组质粒IPSE/pET32a(+)经IPTG诱导后表达出相对分子质量为35700的融合蛋白。纯化的融合蛋白Trx-IPSE在变性或复性条件下均与日本血吸虫病患者混合血清呈阳性反应。Trx-IPSE蛋白也与健康人血清中的非特异性IgE发生反应。
已合成曼氏血吸虫IPSE基因，重组体可与抗日本血吸虫天然抗体IgG反应，并与IgE抗体非特异性结合。
