弓形虫信号转导蛋白14-3-3的高效表达及免疫学诊断的初探

目的　构建弓形虫信号转导蛋白 14 -3- 3基因的重组质粒 ,并在E .coli中高效表达 ,用于免疫学诊断。方法　以限制性内切酶EcoRI和XhoI双酶切质粒pGEM T/Toxo 14 - 3- 3,获得弓形虫信号转导蛋白 14 - 3 -3编码基因片段 ,插入载体pET2 8a ,转化E .coliBL2 1,IPTG诱导表达 ,在非变性条件下纯化融合的表达产物 ,通过Westernblot和ELISA检测其特异的免疫反应性。结果　所构建的弓形虫信号转导蛋白 14 -3 -3(rToxo 14 - 3- 3)在原核系统中的重组表达质粒 ,以 6个His融合蛋白的形式得到高效表达 ,其表达量占细菌裂解液中总蛋白量的 4 9 .2 %。该重组抗原经纯化能被弓形虫感染的人血清所识别。用rToxo 14- 3 -3作为抗原 ,ELISA检测弓形虫病具有高度的敏感性和特异性。结论　rToxo 14 - 3- 3在大肠杆菌中已得到高效表达 ,该重组抗原能有效检测弓形虫的感染 ,可用于弓形虫病诊断试剂盒的研制。

High Expression of Signaling Protein 14-3-3 of Toxoplasma gondii and Its Application in Immunological Diagnosis

Objective To construct a recombinant plasmid with signaling protein 14-3-3 gene of Toxoplasma gondii,express the protein in E.coli and study its application in immunological diagnosis.Methods Digest pGEN-T/Toxo-14-3-3 with EcoR I and Xho I to obtain the signaling protein 14-3-3 gene of Toxoplasma gondii.Insert the gene into vector pET28a,transform to E.coli BL21 and express under induction of IPTG. Purify the expressed fusion protein and detect its specific immunoreactivity by Western blot and ELISA.Results The signaling protein 14-3-3 of Toxoplasma gondii(rToxo-14-3-3) was highly expressed. The expressed product existed in the form of fusion protein,contained 49.2% of total somatic protein,and was recognized by Toxoplasma gondii-infected human serum. The rToxo-14-3-3 as an antigen showed high sensitivity and specificity in the detection of toxoplasmosis by ELISA.Conclusion rToxo-14-3-3 was highly expressed in E.coli. The expressed recombinant antigen was effective in the detection of Toxoplasma gondii infection and might be used for the preparation of diagnostic kit for toxoplasmosis.