蜱传脑炎病毒TBE-E-D3抗原的原核表达纯化及ELISA方法鉴定

摘要

Objective: To obtain a better specific antigen TBE-E-D3 of tick-bome encephalitis virus for clinical diagnostic research, the prokaryotic expression plasmid GSTpET-30a( + )/TBE-E-D3 was constructed, and the His- tag fusion protein His-TBE-E-D3 was induced and purified by affinity chromatography. Methods: The primers were designed and synthesized, and the target gene sequences were amplified by PCR, and it was inserted into GSTpET-30a(+). After clone selection and sequencing, the correct recombinant plasmid of GSTpET-30a( + )/TBE- E-D3 was transferred into E.coli BL21(DE3). The expressed protein was purified by affinity chromatography using Ni-NTA agarose, and the purity level was analyzed by SDS-PAGE. The specificity and sensitivity of recombinant antigen were identified by ELISA. Results: The TBE-E-D3 cDNA was inserted into the vector GSTPET-30a (+) with correct open read frame, and the expression of His-TBE-E-D3 could be induced by adding IPTG. Using the His-TBE-E-D3 protein as antigens to detect samples of patient sera by ELISA, 10 of 15 sera from patients were positive, and 39 of 40 healthy sera were negative. Conclusion: The His-TBE-E-D3 protein expressed in E.coli cells retains good antigenicity, and can be used as candidate antigens for tick-bome encephalitis virus specific serodiagnosis.%目的:构建蜱传脑炎病毒TBE-E-D3抗原的融合蛋白原核表达质粒GSTpET-30a(+)/lBE-E-D3,在大肠杆菌中诱导表达并用亲和层析法纯化,以获得特异性诊断抗原.方法:根据目的基因序列设计PCR引物,利用基因克隆技术构建重组质粒,转入大肠杆菌DH5α后经酶切鉴定,将测序正确的重组质粒转入大肠杆菌BL21 (DE3),IPTG诱导表达后进行亲和层析法纯化,SDS-PAGE分析其表达情况和纯度,间接ELISA法鉴定重组蛋白的特异性和灵敏度.结果:TBE-E-D3基因目的片段以正确的读框插入载体GSTpET-30a(+),通过IPTG诱导在大肠杆菌中正确表达,亲和纯化得到较高纯度的蛋白质；间接ELISA法证明抗原特异性和灵敏度良好,送检的15份阳性患者血清样本中检出10份阳性结果,40份健康人血清样本中检出1份假阳性结果.结论:获得的His-TBE-E-D3融合蛋白具有良好的抗原性,可作为森林脑炎病毒特异性血清学诊断的备选抗原之一.