日本血吸虫TβRII样基因胞外段的克隆、表达及初步鉴定

Cloning,expression and preliminary identification of the TβRII-like extracellular fragment protein from Schistosoma japonicum

目的获得日本血吸虫TGF-β受体II(TβRII)样基因的cDNA完整序列,克隆、原核表达其胞外段蛋白序列,并进行初步免疫学分析。方法应用RACE方法获得SjTβRII样基因cDNA全长序列,构建其胞外段(即N端)原核表达载体,在大肠埃希菌中IPTG诱导表达,表达产物行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。纯化重组蛋白并免疫大鼠制备抗血清,应用Western Blotting方法初步鉴定重组蛋白。结果获得日本血吸虫SjTβRII样基因的cDNA全长序列,为2283bp,编码760个氨基酸,其中胞外段长531bp,编码177个氨基酸。构建的重组质粒在大肠埃希菌中成功表达,经亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别,表明其具有免疫原性和抗原性。结论首次获得日本血吸虫TβRII样基因的cDNA全长序列,其胞外段可在原核表达系统中获得具有免疫原性和抗原性的高效表达,为进一步研究该蛋白的功能奠定了基础。

To study the SjTβRII-like gene from Schistosoma japonicum （S. japonicum）, the coding region of SjTβRII-like was obtained by RACE and the extracellular fragment of SjTβRII-like gene was cloned into the prokaryotic expression vector pET28a （＋） then expressed in E.coli by IPTG induction. The recombinant protein was detected by SDS-PAGE. SD rats were immunized with the purified recombinant protein in order to obtain the antisera and analyze its immunogenicity and antigenicity by Western blotting.It was found that the encoding sequence of the novel SjTβRII-like gene was 2283 bp （encoding 760 amino acids）. Recombinant expression plasmid was successfully constructed, which could elicit specific antibodies efficiently in SD rats, indicating its immunogenicity and antigenicity. Results suggested that the extracelluar fragment of the novel SjTβRII-like gene of S. japonicum could be expressed in the prokaryotic system with immunological and antigenic activities efficiently.