| 摘要: |
| 【目的】 克隆H9N2亚型禽流感病毒(AIV)河南株核蛋白(NP)基因，并将NP主要抗原表位区在大肠杆菌中进行表达，为禽流感基因工程诊断抗原的制备和开发奠定基础。【方法】 根据GenBank公布的禽流感病毒NP基因序列设计引物，采用反转录 聚合酶链反应(RT-PCR)技术，从H9N2亚型AIV 河南株感染的鸡胚尿囊液RNA中扩增AIV河南株NP全基因，将扩增的PCR产物克隆到pGEM-Teasy载体并进行测序。通过PCR和酶切方法将NP主要抗原表位区亚克隆到原核表达载体pET-28a中，构建重组表达质粒pET-28NP，转化大肠杆菌BL21（DE3），在IPTG诱导下表达重组蛋白。【结果】 测序结果表明，克隆的H9N2亚型AIV河南株NP基因全长为1 497 bp，包含1个完整读码框，编码498个氨基酸，与GenBank中不同来源、不同亚型的其他7株禽流感病毒NP基因的核苷酸同源性达到95%以上，氨基酸同源性达到98%以上。SDS-PAGE可检测到相对分子质量约为42 ku的重组蛋白。经1.0 mmol/L IPTG诱导5 h时，重组蛋白的表达量最高。Western blot分析表明，重组蛋白能够与H9亚型AIV阳性血清发生特异反应。【结论】NP基因非常保守，建立的表达系统能够表达NP蛋白且表达蛋白具有良好的免疫原性。 |
| 关键词: 禽流感病毒 河南株 核蛋白 克隆 表达 |
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| 基金项目:国家“十一五”科技支撑计划专项（2006BAD06A08） |
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| Cloning and expression of NP gene of H9N2 subtype avian influenza virus henan isolate in Escherichia coli |
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| Abstract: |
| 【Objective】 The research was to study cloning and expression of nucleoprotein (NP) gene of H9N2 subtype avian influenza virus (AIV) henan isolate in Escherichia coli.【Method】 A pair of specific primers were designed and synthesized according to NP gene sequences published in GenBank. NP cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the virus RNA directly extracted from chicken embryo allantoic fluid infected by H9N2 subtype AIV henan isolate,and cloned into pGEM-T easy vector and sequenced.The NP gene segment which encoded the major epitopes of nucleoprotein was subcloned into prokaryotic expression vector pET-28a(+) using PCR and enzyme digestion.The resulting plasmid,pET-28 NP ,was used to transform into E.coli BL21(DE3),which can highly express recombinant NP protein by using IPTG induction.【Result】 The results showed that the nucleotide sequence of NP gene was 1497 bp in length,which included one open reading frame,encoding 498 amino acid residues.When the nucleotide sequence of NP gene of henna isolate was compared with that of 7 strains of AIV in GenBank,homology was higher than 95% at nucleotide level,and the deduced amino acid homology was higher than 98%.The expressed product was identified by SDS PAGE and Western blot.The results showed that recombinant protein was about 45 ku,and can highly be expressed in E.coli BL21(DE3) by using 1.0 mmol/L IPTG induction for 5 h.The recombinant protein can react with sera against H9 subtype AIV.【Conclusion】 NP gene for H9N2 AIV was highly reserved.Recombinant NP protein for H9N2 AIV can be expressed in expression system pET-28NP and had good immunogenicity. |
| Key words: avian influenza virus henna isolate nucleoprotein cloning expression |
