布鲁氏菌鞭毛钩蛋白FlgE和融合蛋白BLS-FlgE的克隆、表达及免疫诊断研究

目的 分别构建含目的基因flgE和bls-flgE的原核表达载体,表达纯化重组蛋白,并对其免疫诊断价值进行初步评价。方法 从NCBI得到布鲁氏菌鞭毛钩蛋白基因flgE和2,4二氧四氢蝶啶合酶基因(bls)序列,设计引物,利用PCR技术扩增得到目的基因,与载体pET30a连接,转化E.coli BL21感受态细胞,IPTG诱导蛋白表达,SDS-PAGE及Western blot鉴定,使用带His标签的蛋白纯化试剂盒纯化蛋白。纯化后的蛋白建立ELISA法检测布病阳性血清和健康人血清,收集灵敏度和特异度数据。结果 成功克隆了FlgE鞭毛钩蛋白和BLS-FlgE融合蛋白,经过优化表达条件,目的蛋白获得较大量的表达,免疫印迹结果显示可被阳性病人血清识别。以FlgE蛋白构建ELISA法检测得到灵敏度和特异度分别为70.83%和41.67%,以BLS-FlgE蛋白构建ELISA法检测得到灵敏度和特异度分别为68.75%和52.08%。结论 成功表达布鲁氏菌鞭毛钩蛋白FlgE和融合蛋白BLS-FlgE,分别作为诊断抗原总体符合率低,免疫诊断价值较低。

Cloning,expression and immunodiagnostic research on the Brucella flagellin hook protein FlgE and fusion protein BLS-FlgE

The Brucella flagellin gene flgE and 2,4 dioxytetrahydrodiopterin synthase gene (bls) sequences were obtained from NCBI. Primers were designed, and PCR was used to amplify the target gene, ligate it into the vector pET30a and transform E. coli BL21 competent cells. IPTG was used to induce protein expression, and SDS-PAGE and western blotting were performed. A His-tagged protein purification kit was used to purify the protein. The purified protein was used in ELISA analysis to detect Brucellosis-positive serum and healthy human serum. The sensitivity and specificity were assessed. The flagellin FlgE and BLS-FlgE fusion proteins were successfully cloned. After optimizing the expression conditions, the target protein was expressed in a large amount. The results of immune blotting showed that it could be recognized by the serum of positive patients. The sensitivity and specificity of FlgE protein constructed ELISA method were 70.83% and 41.67%, and the sensitivity and specificity of BLS-FlgE protein constructed ELISA method were 68.75% and 52.08%. The two proteins had a low overall coincidence rate and poor immunodiagnostic value as diagnostic antigens.