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目的克隆截短的弓形虫表面抗原SAG2C基因,在大肠埃希菌中表达SAG2C蛋白,并探讨其在弓形虫病诊断中的应用。方法对已知的弓形虫SAG2C基因序列进行部分取舍,用RT-PCR技术从弓形虫Prugniaud(PRU)株的总RNA中扩增截短的SAG2基因片段,插入载体pET32a(+)中,转化大肠埃希菌BL21,IPTG诱导表达,应用Western blot和ELISA检测重组表达蛋白的免疫反应性。用重组SAG2C蛋白ELISA法检测弓形虫感染血清特异抗体,观察初步应用效果。结果从弓形虫PRU株总RNA中扩增出截短的SAG2C基因片段,成功构建了重组表达质粒pET32a(+)-tSAG2C;该重组质粒经IPTG诱导能表达可溶性大小为51ku的SAG2C蛋白。Western blot显示重组SAG2C能被弓形虫感染小鼠血清识别;以重组SAG2C蛋白、重组SAG1蛋白及BAG1蛋白ELISA检测精神病患者血清弓形虫抗体,阳性率分别为8.07%(23/285)、4.56%(13/285)和7.37%(21/285),差异无统计学意义(P>0.05)。结论成功构建了重组质粒pET32a(+)-tSAG2C,表达的融合蛋白具有免疫反应性,具有用于弓形虫感染诊断的潜在价值。
Objective To clone the truncated Toxoplasma gondii surface antigen SAG2C gene and express SAG2C protein in Escherichia coli and to explore its application in the diagnosis of toxoplasmosis. Methods The sequence of SAG2C gene of Toxoplasma gondii was partially subtracted. The truncated SAG2 gene fragment was amplified from the total RNA of Toxoplasma gondii Prugniaud (PRU) by RT-PCR and inserted into the vector pET32a (+) to transform into the large intestine Escherichia coli BL21, IPTG induced expression, Western blot and ELISA detection of recombinant protein expression of immunoreactivity. The recombinant SAG2C protein was used to detect Toxoplasma gondii infection with serum specific antibody and the preliminary application effect was observed. Results The truncated SAG2C gene fragment was amplified from the total RNA of Toxoplasma gondii PRU strain. The recombinant plasmid pET32a (+) - tSAG2C was constructed successfully. The soluble recombinant SAG2C protein was induced by IPTG. Western blot showed that recombinant SAG2C could be recognized by the serum of Toxoplasma gondii-infected mice. The positive rate of Toxoplasma gondii antibody was 8.07% (23/285) and 4.56% (P <0.05) by using recombinant SAG2C protein, recombinant SAG1 protein and BAG1 protein ELISA, 13/285) and 7.37% (21/285) respectively, with no significant difference (P> 0.05). Conclusion The recombinant plasmid pET32a (+) - tSAG2C was constructed successfully. The expressed fusion protein is immunoreactive and has the potential value for the diagnosis of Toxoplasma gondii infection.