链球菌蛋白G(streptococcal protein G,SPG)具有与多种动物IgG结合的特性。本研究对链球菌蛋白G进行生物学分析,设计重组蛋白G的基因序列,构建重组蛋白G的原核表达质粒,并在大肠杆菌中进行表达,得到带有GST标签的融合蛋白,通过GST亲和...链球菌蛋白G(streptococcal protein G,SPG)具有与多种动物IgG结合的特性。本研究对链球菌蛋白G进行生物学分析,设计重组蛋白G的基因序列,构建重组蛋白G的原核表达质粒,并在大肠杆菌中进行表达,得到带有GST标签的融合蛋白,通过GST亲和层析纯化蛋白。Western blot结果显示,重组蛋白具备与IgG结合的能力。ELISA测定重组蛋白与牛、山羊、兔、鼠4个物种IgG的亲和力,显示重组蛋白G的亲和力普遍高于商品化链球菌蛋白G。本研究成功重构并表达、纯化了链球菌重组蛋白G,得到其与4个物种抗体IgG的亲和力常数,有助于SPG在其他相关研究中的应用。展开更多
Background:Schistosomiasis remains a major public health concern in China and an epidemiological survey has revealed that schistosome-infected bovines and goats are the main transmission sources for the disease.Theref...Background:Schistosomiasis remains a major public health concern in China and an epidemiological survey has revealed that schistosome-infected bovines and goats are the main transmission sources for the disease.Therefore,development of a sensitive technique for the diagnosis of schistosomiasis in domestic animals is necessary.Method:A novel colloidal gold immunochromatography assay(GICA)strip was developed for detecting Schistosoma japonicum in domestic animals.The colloidal gold was conjugated with recombinant streptococcal protein G(rSPG).As the test and control lines,the schistosome soluble egg antigen and rSPG,respectively,were blotted on nitrocellulose membrane.Results:The lowest detectable serum dilution was 1∶640 for schistosome-infected buffaloes.The cross-reaction rate of GICA was 14.29%with Paramphistomum sp.in buffaloes,16.67%with Haemonchus sp.in goats,and 33.33%with Orientobilharzia sp.in goats.These results were slightly lower and similar to those obtained through ELISA.Moreover,the strips for detecting S.japonicum in mice,rabbits,buffaloes,and goats showed high sensitivity(100.00%,100.00%,100.00%,and 100.00%,respectively)and specificity(100.00%,100.00%,94.23%,and 88.64%,respectively).And the sensitivity or specificity of the GICA strips did not present any significant differences after storage for 12 months at room temperature.When compared with ELISA,the GICA strips exhibited similar sensitivity and specificity in the diagnosis of schistosomiasis in mice,rabbits,buffaloes,and goats.Besides,only 5μl of serum are required for the test and the detection can be completed within 5 min.Conclusion:This study is the first to develop a GICA strip using gold-rSPG conjugate for the diagnosing of schistosomiasis in domestic animals,and preliminary results showed that the developed strip may be suitable for large-scale screening of schistosomiasis in endemic areas.展开更多
文摘链球菌蛋白G(streptococcal protein G,SPG)具有与多种动物IgG结合的特性。本研究对链球菌蛋白G进行生物学分析,设计重组蛋白G的基因序列,构建重组蛋白G的原核表达质粒,并在大肠杆菌中进行表达,得到带有GST标签的融合蛋白,通过GST亲和层析纯化蛋白。Western blot结果显示,重组蛋白具备与IgG结合的能力。ELISA测定重组蛋白与牛、山羊、兔、鼠4个物种IgG的亲和力,显示重组蛋白G的亲和力普遍高于商品化链球菌蛋白G。本研究成功重构并表达、纯化了链球菌重组蛋白G,得到其与4个物种抗体IgG的亲和力常数,有助于SPG在其他相关研究中的应用。
基金This study was financially supported by the Basic Foundation for Scientific Research of State-level Public Welfare Institutes of China(Grant no.2014JB01).
文摘Background:Schistosomiasis remains a major public health concern in China and an epidemiological survey has revealed that schistosome-infected bovines and goats are the main transmission sources for the disease.Therefore,development of a sensitive technique for the diagnosis of schistosomiasis in domestic animals is necessary.Method:A novel colloidal gold immunochromatography assay(GICA)strip was developed for detecting Schistosoma japonicum in domestic animals.The colloidal gold was conjugated with recombinant streptococcal protein G(rSPG).As the test and control lines,the schistosome soluble egg antigen and rSPG,respectively,were blotted on nitrocellulose membrane.Results:The lowest detectable serum dilution was 1∶640 for schistosome-infected buffaloes.The cross-reaction rate of GICA was 14.29%with Paramphistomum sp.in buffaloes,16.67%with Haemonchus sp.in goats,and 33.33%with Orientobilharzia sp.in goats.These results were slightly lower and similar to those obtained through ELISA.Moreover,the strips for detecting S.japonicum in mice,rabbits,buffaloes,and goats showed high sensitivity(100.00%,100.00%,100.00%,and 100.00%,respectively)and specificity(100.00%,100.00%,94.23%,and 88.64%,respectively).And the sensitivity or specificity of the GICA strips did not present any significant differences after storage for 12 months at room temperature.When compared with ELISA,the GICA strips exhibited similar sensitivity and specificity in the diagnosis of schistosomiasis in mice,rabbits,buffaloes,and goats.Besides,only 5μl of serum are required for the test and the detection can be completed within 5 min.Conclusion:This study is the first to develop a GICA strip using gold-rSPG conjugate for the diagnosing of schistosomiasis in domestic animals,and preliminary results showed that the developed strip may be suitable for large-scale screening of schistosomiasis in endemic areas.
