| 猪繁殖与呼吸综合征病毒ORF7基因的克隆、高效表达与鉴定 |
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| 引用本文： | 张秀云,吴斌,肇慧君,贾赟,刘霞,张瑞. 猪繁殖与呼吸综合征病毒ORF7基因的克隆、高效表达与鉴定[J]. 大连工业大学学报, 2011, 30(1): 18-22 |
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| 作者姓名： | 张秀云 吴斌 肇慧君 贾赟 刘霞 张瑞 |
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| 作者单位： | 张秀云,ZHANG Xiu-yun(大连工业大学生物工程学院,辽宁大连,116034);吴斌,肇慧君,贾赟,刘霞,WU Bin,ZHAO Hui-jun,JIA Yun,LIU Xia(辽宁出入境检验检疫局,辽宁大连,116001);张瑞,ZHANG Rui(河南农业大学牧医工程学院,河南郑州,450002) |
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| 摘 要： | 根据猪繁殖与呼吸综合症病毒(PRRSV)VR2332株ORF7的全长基因序列设计一对特异性引物,应用RT-PCR扩增出完整的ORF7基因,并将PCR产物克隆至pMD-18T载体进行测序分析.双酶切测序后重组质粒pMD-18T-N得到目的片段连接至原核表达载体pET-28a(+)上,构建重组质粒pET-28a(+)-N....
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| 关 键 词： | 猪繁殖与呼吸综合征病毒(PRRSV) 核衣壳蛋白基因(ORF7) 基因克隆 原核表达 |
Cloning, expression and identification of porcine reproductive and respiratory syndrome virus ORF7 |
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| ZHANG Xiu-yun,WU Bin,ZHAO Hui-jun,JIA Yun,LIU Xia,ZHANG Rui. Cloning, expression and identification of porcine reproductive and respiratory syndrome virus ORF7[J]. Journal of Dalian Dalian Polytechnic University, 2011, 30(1): 18-22 |
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| Authors: | ZHANG Xiu-yun WU Bin ZHAO Hui-jun JIA Yun LIU Xia ZHANG Rui |
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| Affiliation: | 1.School of Bioengineering,Dalian Polytechnic University,Dalian 116034,China;2.Liaoning Entry-Exit Inspection and Quarantine Bureau,Dalian 116001,China;3.Engineering College of Animal Husbandry and Veterinary Science,Henan Agricultural University,Zhengzhou 450002,China |
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| Abstract: | A pair of specific primers was designed according to gene sequences ORF7 of PRRSV VR2332 strain to amplify a complete ORF7 gene by RT-PCR.The PCR products were cloned into the vector pMD-18T and sequenced.The double-digested fragment of the sequenced recombinant plasmid pMD-18T-N was obtained and connected to prokaryotic expression vector pET-28a(+).Recombinant plasmid pET-28a(+)-N was converted to the host bacteria BL21 and the conditions(concentrations of IPTG,induction time,induction temperature) were optimized.The results showed that 37 ℃,0.2 mmol/L IPTG induced 5 h can realize high expression of the recombinant protein.Ni-NTA affinity chromatography and Western-blotting analysis showed that expression recombinant N protein can react specifically with PRRSV-positive sera. |
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| Keywords: | porcine reproductive and respiratory syndrome virus(PRRSV) nucleocapsid protein gene(ORF7) gene cloning prokaryotic expression |
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