论文部分内容阅读
目的原核表达弓形虫棒状体ROP21基因,并对rROP21蛋白进行免疫学鉴定。方法根据已发表的ROP21基因序列设计引物,通过RT-PCR方法扩增弓形虫RH株的ROP21基因。将ROP21基因克隆到原核表达载体pET-28a(+)后转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导表达目的蛋白,纯化后进行SDS-PAGE分析、Western blot及IFA分析。结果经PCR和酶切鉴定,成功构建重组表达载体。重组质粒转化BL21(DE3)后经IPTG诱导,重组蛋白(rROP21)以包涵体形式高效表达,分子质量单位约为45ku。Western blot显示重组rROP21蛋白能被his标签抗体及弓形虫病患者血清识别。用rROP21免疫小鼠,获得滴度为1∶104的抗体血清。IFA显示弓形虫速殖子体内存在ROP21蛋白。结论构建的重组质粒pET28a-ROP21表达的弓形虫rROP21具有抗性,可作为血清学诊断候选抗原,为进一步研究其生物学功能奠定了基础。
Objective To prokaryotic express the Toxoplasma gondii ROP21 gene and to identify the rROP21 protein. Methods According to the published sequence of ROP21 gene, primers were designed and the ROP21 gene of Toxoplasma gondii RH strain was amplified by RT-PCR. The ROP21 gene was cloned into the prokaryotic expression vector pET-28a (+) and transformed into E. coli BL21 (DE3) competent cells. IPTG induced expression of the target protein, purified by SDS-PAGE analysis, Western blot and IFA analysis. Results PCR and restriction enzyme digestion, the successful construction of recombinant expression vector. Recombinant plasmid was transformed into BL21 (DE3) and induced by IPTG. Recombinant protein (rROP21) was highly expressed in inclusion bodies with the molecular mass of about 45ku. Western blot showed that the recombinant rROP21 protein was recognized by his-tagged antibodies and the serum of toxoplasmosis patients. Mice were immunized with rROP21 to obtain antibody sera with a titer of 1: 104. IFA shows the presence of ROP21 protein in Toxoplasma gondii tachyzoites. Conclusion The recombinant plasmid pET28a-ROP21 expressed in E.coli rROP21 was resistant and could be used as a candidate antigen for serodiagnosis, which laid the foundation for further study of its biological functions.