禽流感病毒核蛋白基因的克隆及在大肠杆菌中的表达

目的　以 1株H5N1 亚型禽流感病毒RNA为模板扩增NP全基因cDNA ,并在大肠杆菌中进行表达。方法　提取禽流感病毒RNA ,以RT PCR法扩增编码NP基因cDNA并测序 ,将其克隆到pET 2 8a原核表达载体 ,并在大肠杆菌BL2 1(DE3)plysS中表达 ,用SDS PAGE和Westernblot检测表达产物。结果　所克隆的核苷酸片段包含了核蛋白基因完整阅读框架 ,编码 4 98个氨基酸 ,构建的重组表达载体在大肠杆菌BL2 1(DE3)plysS中表达出相对分子质量约为 5 5 0 0 0的重组蛋白 ,该重组蛋白具有良好的免疫原性。结论　已成功克隆和表达了禽流感病毒核蛋白基因 ,为禽流感新型免疫学诊断试剂的研制奠定了基础

Cloning and Expression of Nucleoprotein (NP)Gene of Avian Influenza Virus in E.Coli

Objective To amplify the cDNA encoding complete nucleoprotein(NP)gene from a strain of avian influenza virus isolate(H_5N_1) and express the gene in E.coli.Methods Extract RNA from H_5N_1 strain and amplify the cDNA encoding complete NP gene by RT-PCR.Identify the amplified NP gene by sequencing, insert into prokaryotic expression vecotr pET-28a and transform to E.coli BL21(DE3)plys S for expression.Detect the expressed protein by SDS-PAGE and Western blot.Results The cloned cDNA fragments covered the whole open reading frame of NP gene and encoded 498 amino acids.The expressed recombinant protein, with a relative molecular weight of about 55 000, showed good immunogenicity.Conclusion The NP gene of avian influenza virus was successfully cloned and expressed.The study laid a foundation of development of a novel immunological diagnostic kit for avian influenza.