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
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摘要
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.
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.
引文
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[2] 张成芳,张海国,李瑾.弓形虫病免疫学诊断的研究进展[J].中华诊断学电子杂志,2017,5(1):59-61.
[3] Allahyari M,Mohabati R,Babaie J,et al.Production of in-vitro refolded and highly antigenic SAG 1 for development of a sensitive and specific Toxoplasma IgG ELISA[J].Immunol Methods,2015(416):157-66.
[4] 许汝槿,夏建萍,黄建锋,等.杭州市高危人群弓形虫感染血清流行病学调查[J].中国血吸虫病防治杂志,2017,29(6):756-60.
[5] Burg JL,Perelman D,Kasper LH,et al.Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii[J].Immunol,1988(141):3584-91.
[6] Wang Y,Yin H.Research progress on surface antigen 1 (SAG1) of Toxoplasma gondii[J].Parasit Vectors,2014(7):180.
[7] Steinberg HE,Russo P,Angulo N,et al.Toward detection of toxoplasmosis from urine in mice using hydro-gel nanoparticles concentration and parallel reaction monitoring mass spectrometry[J].Nanomedicine,2018,14(2):461-9.
[8] Hegazy S,Farid A,Rabae I,et al.Novel IMB-ELISA assay for rapid diagnosis of human toxoplasmosis using SAG1 antigen[J].Jpn J Infect Dis,2015,68(6):474-80.
[9] Khanaliha K,Motazedian M H,Kazemi B,et al.Evaluation of recombinant SAG1,SAG2,and SAG3 antigens for serodiagnosis of toxoplasmosis[J].Kor J Parasitol,2014,52(2):137-42.
[10] Tkachenko H,Xie D,Coleman,et al.Multifunctional gold nanoparticle-peptide complexesfor nuclear targeting[J].J Am Chem Soc,2003,125(16):4700-1.
[11] Zhang X,Young M,Lyandres A,et al.Rapid detection of an anthrax biomarker by surface-enhanced Raman spectroscopy[J].J Am Chem Soc,2005,127(12):4484-9.
[12] Cheng M C,Cuda G,Bunimovich Y L,et al.Nanotechnologies for biomolecular detection and medical diagnostics[J].Curr Opin Chem Biol,2006,10(1):11-9.
[13] 孙文霞,袁仕善,黄昊文,等.结核分枝杆菌抗原模拟肽纳米金生物传感器诊断结核病[J].中国实验诊断学,2012,16(12):2162-04.