日本血吸虫弹性蛋白酶基因的克隆、表达及虫期特异性转录

Cloning, Expression of Schistosoma japonicum Elastase Gene and its Stage-specific Transcription

目的克隆和表达日本血吸虫弹性蛋白酶基因(SjCE-2b),纯化表达重组蛋白,并研究该基因在各虫期的转录情况。方法预测SjCE-2b基因的编码序列。绘制血吸虫尾蚴弹性蛋白酶家族成员系统进化树。用RT-PCR和蛋白质印迹(Western blotting)分析SjCE-2b基因在日本血吸虫各期转录的差异。将RT-PCR扩增得到的SjCE-2b基因亚克隆至载体pET28b,在大肠埃希菌中诱导表达得到重组蛋白rSjCE-2b,用组氨酸标签亲和层析法纯化表达产物,Western blotting分析其免疫原性。结果在虫卵、子胞蚴和成虫检测到SjCE-2b基因转录本(714bp),表达的重组蛋白rSjCE-2b,相对分子质量约为Mr 31000,与预测的融合蛋白相对分子质量相符。构建了重组原核表达载体SjCE-2b/pET28b,并在大肠埃希菌中表达。纯化后的重组蛋白rSjCE-2b可被日本血吸虫感染的兔血清识别。结论在日本血吸虫虫卵、子胞蚴和成虫发现SjCE-2b基因转录本。SjCE-2b基因有可能成为潜在的日本血吸虫病疫苗候选抗原和药物及诊断靶点。

Objective To clone, express and purify Schistosoma japonicum elastase-2b gene（SjCE-2b）, and ana- lyze its stage-specific transcription, and expression. Methods The coding sequence of the Sj gene was predicted, and a phylogenetic tree of Sj elastase was drawn. RT-PCR and Western blot were used to investigate the differential transcription and expression of SjCE-2b gene during the developmental stages. The SjCE-2b gene obtained by RT-PCR was subcloned into pET28b, and expressed in E.coli （rSjCE-2b）. The expressed protein was purified with His.Tag affinity chromatogra- phy. Western blotting was used to investigate the immunogenicity. Results RT-PCR showed specific bands in sporocysts, eggs and adult worms, while Western blot showed that the recombinant protein（rSjCE-2b） existed only in cercariae and sporosysts, with Mr 31 000. The expression vector of SjCE-2b/pET28b was constructed and expressed in E. coli. The recombinant protein rSjCE-2b was specifically recognized by the S.japonicum-infected rabbit serum. Conclusion The transcript of S.japonicum elastase-2b gene was found in sporoeysts, eggs and adult worms, and this gene might be a potential candidate for vaccine, for drug and diagnosis target.