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目的将乙型脑炎病毒NS1蛋白基因克隆至pET28-a(+)表达载体,构建原核表达载体pET-NS1,并使该基因在E.coli中高效表达,为进一步研制乙脑早期诊断试剂奠定基础。方法根据GenBank中提供的乙型脑炎病毒SA14-14-2株全基因序列设计引物,通过反转录及巢式PCR方法扩增目的片段,测序后连接pET28-a(+)载体,构建重组表达载体pET-NS1,转化大肠杆菌BL21(DE3)后,利用IPTG诱导获得高效表达。结果扩增出了1300bp的基因片段,与预期大小一致,测序后经Blast验证与已发表乙型脑炎病毒SA14-14-2株NS1蛋白基因序列同源性为100%,成功克隆至表达载体pET28-a(+)并获得高效表达,表达产物分子量约为45kD,Western blot分析表明该表达产物具有良好抗原性。结论该表达产物的稳定高效表达及其抗原特异性为乙脑的诊断试剂开发提供了依据。
Objective To clone the NS1 protein of Japanese encephalitis virus into the expression vector pET28-a (+) and construct the prokaryotic expression vector pET-NS1, and to express the gene in E.coli efficiently and lay a foundation for further development of early diagnostic reagent for Japanese encephalitis basis. Methods According to the sequence of complete genome of Japanese encephalitis virus SA14-14-2 provided in GenBank, primers were designed and amplified by reverse transcription and nested PCR. After sequencing, pET28-a (+) vector was ligated to construct recombinant The expression vector pET-NS1 was transformed into E. coli BL21 (DE3) and then induced by IPTG. Results The gene fragment of 1300bp was amplified and was consistent with the expected size. After sequencing, the sequence identity of NS1 protein of SA14-14-2 strain was 100% verified by Blast and successfully cloned into the expression vector pET28-a (+) was obtained and expressed efficiently. The molecular weight of the expressed product was about 45 kD. Western blot analysis showed that the expressed product had good antigenicity. Conclusion The stable and efficient expression of the expressed product and its antigen specificity provide the basis for the development of diagnostic reagents for JE.