应用弓形虫截短型SAG1纳米金免疫传感器检测弓形虫抗体的研究

Detection of Toxoplasma gondii antibodies with a gold nanorod immunosensor using truncated SAG1 as an antigen

目的克隆表达弓形虫主要表面抗原1截短型片段(tSAG1),建立tSAG1纳米金免疫传感器,用于检测弓形虫抗体。方法 PCR扩增tSAG1基因片段并克隆至pMD18-T载体,测序鉴定后亚克隆至表达载体pET-28a(+),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达tSAG1,金属螯合层析法进行纯化,Western blot分析其免疫活性;采用种子生长法制备金纳米棒,偶联tSAG1,构建纳米金免疫传感器,用于检测人血清弓形虫抗体,计算其敏感度、特异度、阳性及阴性预测值和诊断效率。结果构建了重组表达质粒pET-28a (+)-tSAG1,表达纯化获得具有反应原性的tSAG1,其相对分子质量约为30×10^3。用制备的tSAG1纳米金免疫传感器检测人血清弓形虫抗体的敏感度、特异度、阳性预测值、阴性预测值及诊断效率分别为80.9%、90.0%、90.2%、80.6%和85.2%。结论基于重组tSAG1的纳米金免疫传感器可用于弓形虫抗体的检测。

Objective To clone and express the truncated major surface antigen 1(tSAG1) from Toxoplasma gondii and create a gold nanorod immunosensor to detect antibodies to tSAG1 from T. gondii. Methods The tSAG1 gene fragment was amplified using PCR and cloned into the vector pMD18-T. After it was verified as correct using DNA sequencing, the tSAG1 gene fragment was subcloned into the expression vector pET-28 a(+). Expression of tSAG1 was induced with isopropyl-β-D-thiogalactoside(IPTG) in E. coli BL21 containing the recombinant plasmid pET-28 a(+)-tSAG1, and tSAG1 was purified using metal chelate chromatography. The immunological activity of tSAG1 was analyzed using Western blotting. Gold nanorods were prepared by seed growth and coupled with tSAG1 to construct a gold nanorod immunosensor. Antibodies to tSAG1 of T. gondii in human serum were detected using the gold nanorod immunosensor, and the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of the gold nanorod immunosensor were calculated. Results The recombinant expression plasmid pET-28 a(+)-tSAG1 was constructed. The tSAG1 expressed in E. coli BL21 was purified using metal chelate chromatography and reacted to human sera with toxoplasmosis;tSAG1 had a relative molecular mass of about 30×10^3. When diagnosing human toxoplasmosis, the gold nanorod immunosensor had a sensitivity of 80.9%, a specificity of 90.0%, a positive predictive value of 90.2%, a negative predictive value of 80.6%, and a diagnostic efficiency of 85.2%. Conclusion A gold nanorod immunosensor based on recombinant tSAG1 is useful for the detection of antibodies to tSAG1 of T. gondii.