【摘要】 目的对刚地弓形虫peroxiredoxin(TgPrx)基因进行克隆、表达和免疫原性分析。方法收集、纯化RH株弓形虫速殖子,提取总RNA;设计合成引物并引入EcoRI和XhoI酶切位点,RT-PCR扩增编码TgPrx的基因片段克隆到原核质粒pET30a(+)中,经双酶切、PCR及测序鉴定阳性克隆;在大肠杆菌BL21/DE3中用IPTG诱导表达,表达产物经SDS-PAGE进行鉴定,重组蛋白用Western blotting分析其免疫原性。结果从弓形虫RH株cDNA中扩增出591bp的TgPrx基因片段,并成功构建重组质粒pET30a(+)/TgPrx;SDS-PAGE结果表明,目的基因在大肠杆菌BL21/DE3中高效表达。重组蛋白的相对分子量约32kDa,Western blotting显示其能被兔抗弓形虫免疫血清识别。结论RH株刚地弓形虫peroxiredoxin可在原核表达系统中高效表达,该重组蛋白具有免疫原性,有望作为弓形虫疫苗的候选抗原。更多还原

【Abstract】 The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals.Research on developing safe and effective vaccines is the best strategy for the prevention of toxoplasmosis,though not available yet.In this study,the peroxiredoxin gene of T.gondii was cloned and expressed and the immunogenicity of the recombinant protein was analyzed.T.gondii tachyzoites of RH strain were harvested and genomic RNA was prepared.A pair of primers and restriction site EcoRI/XhoI was designed.The coding region of peroxiredoxin was amplified with RT-PCR and cloned into the prokaryotic expression vector pET30a(+).The recombinants were confirmed by EcoRI/XhoI digestion,PCR,and DNA sequencing and then transformed into E.coli BL21 induced by IPTG.The expressed proteins were analyzed by SDS-PAGE,its immunogenicity was analyzed by Western blotting.Result indicated that T.gondii encoding peroxiredoxin gene with a molecular size of 591 bp and the recombinant pET30a/TgPrx was successfully constructed.The results of SDS-PAGE and Western blotting revealed that the molecular weight of the recombinant protein was approximately 32 kDa and could be recognized by rabbit antiserum against toxoplasma.The results suggest that the peroxiredoxin gene of T.gondii has been cloned and expressed,and immunogenicity of the recombinant protein has been confirmed with Western blotting indicating that it may be used as vaccine candidate antigen.更多还原