弓形虫表面抗原P35基因片段的表达纯化和抗原性分析

目的制备具有免疫活性的弓形虫表面抗原P35基因片段的重组蛋白,并对其抗原性进行分析。方法根据弓形虫RH株表面抗原P35的cDNA序列设计一对引物,利用PCR技术从RH株弓形虫基因组中扩增出P35的基因片段,将其克隆到T载体中,并通过基因测序加以证实;将其亚克隆至原核表达载体pET KDO中,在大肠杆菌中经IPTG诱导表达。采用免疫印迹法对表达产物进行抗原性分析。结果从弓形虫RH株基因组中成功扩增到P35目的基因,该基因在原核系统中经诱导表达出分子量约42 000 Da大小的融合蛋白,经免疫印迹实验表明,表达产物具有良好的抗原性,经亲和层析纯化后得到了重组蛋白。结论运用基因工程技术得到了高纯度并具有良好抗原性的弓形虫重组P35融合蛋白。

Expression, purification and antigenicity of P35 gene of Toxoplasma gondii

Objective P35 is an important surface protein for Toxoplasma gondii.To obtain the highly pure and specific antigenicity protein,the gene P35 was cloned and its product was expressed in E.coli Rosetta.The expressed protein was purified and its immunogenecity was studied.Methods A pair of primers was designed according to cDNA sequence of P35,and then the P35 gene amplified by PCR was cloned into the vector pGEM-T and proved by DNA sequencing.The P35 gene was subcloned into prokaryotic expression vector pET-KDO,its expression was induced by IPTG,and the target protein was obtained by affinity chromatography.Results The P35 gene was successfully amplified from genome DNA of Toxoplasma gondii RH strain and a fusion protein was expressed in E.coli.The molecular weight of the expressed protein was about Mr 42 000.Western blotting indicated that the antigenicity of the protein was specific.Conclusions The plasmid pET-KDO-p35 is constructed and the high efficient expression of P35 fusion protein has been achieved in E.coli.The fusion protein shows a specific antigenicity,the P35 fusion protein has a potential value in the diagnosis of toxoplasmosis.