| 摘要: |
| 【目的】构建H9N2亚型禽流感病毒（AIV）NS1基因的原核和真核表达载体，将其分别在大肠杆菌和293细胞中进行表达，为进一步研究NS1蛋白的功能及其与宿主的相互作用，以及鉴定禽流感疫苗免疫禽和野毒感染禽ELISA诊断试剂盒的制备提供理论依据。【方法】用RT-PCR方法扩增H9N2亚型AIV 的NS1基因，将其定向克隆到原核表达载体pET-32a（+）中构建NS1基因的原核重组质粒，转化大肠杆菌BL21（DE3）后，用异丙基硫代半乳糖苷（IPTG）诱导表达；同时，将NS1基因克隆到真核表达载体pEGFP-C1中，构建NS1基因真核重组质粒，将其瞬时转染293细胞，48 h后收集感染细胞,对大肠杆菌表达产物和293细胞表达产物进行Western blotting分析。【结果】成功构建了NS1基因的原核表达载体pET-32a-NS1和真核表达载体pEGFP-C1-NS1。Western-blotting结果表明，大肠杆菌和293细胞的表达产物均能检测到特异性条带（大肠杆菌的表达产物在约44 ku处出现阳性条带，293细胞表达产物在约55 ku处出现特异条带）。【结论】NS1基因在大肠杆菌和293细胞中得到成功表达，获得的NS1蛋白具有良好的抗原活性。 |
| 关键词: H9N2亚型禽流感病毒 NS1基因 大肠杆菌 293细胞 原核表达 真核表达 |
| DOI： |
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| 基金项目:陕西省科技攻关项目（2005K01-G20-2） |
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| Prokaryotic and eucaryotic expression of H9N2 avian influenza virus NS1 gene |
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| Abstract: |
| 【Objective】The study was to construct the prokaryotic expression vector pET-32a-NS1 and eukaryotic expression vector pEGFP-C1-NS1 for the expression of the NS1 gene of H9N2 avian influenza virus in E.coliand 293 cells.【Method】NS1 gene was amplified by RT-PCR.The NS1 gene was cloned directionaly into the pET-32a（+）.The recombinant prokaryotic expression plasmid was constructed and transformed into E.coli BL21(DE3),and induced by IPTG;The NS1 gene was cloned into pEGFP-C1.The recombinant eukaryotic expression plasmid was constructed and transfected into 293 cells by LipofectamineTM 2000,and the transfected cells were collected 48 hours after transfection.Meanwhile,the expressed products were analyzed with Western-blotting.【Result】Results showed that the prokaryotic expression vector and eukaryotic expression vector of NS1 gene were successfully constructed,the special band of NS1 protein expressed in E.coli and 293 cells were identified by Western blotting.【Conclusion】 NS1 gene was successfully expressed in E.coli and 293 cells,Western blotting test showed that the NS1 proteins had very good antigenicity. |
| Key words: H9N2 avian influenza virus NS1 gene E.coli 293 cell prokaryotic expression eucaryotic expression |
