摘要
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(+)/TBE-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。
Objective： To obtain a better specific antigen TBE-E-D3 of tick-borne 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-borne encephalitis virus specific serodiagnosis.
出处
《生物技术通讯》
CAS
2011年第5期636-639,666,共5页
Letters in Biotechnology
基金
国家科技重大专项(2009ZX10601)
关键词
蜱传脑炎病毒
TBE-E-D3抗原
原核表达
纯化
特异性
tick-borne encephalitis virus
TBE-E-D3 antigen
prokaryotic expression
purification
specificity
