H3N2亚型SIV核蛋白基因的克隆及在大肠杆菌中的表达  

Clone of nucleoprotein gene of influenza A virus(H3N2)and its expression in Escherichia coli

作　　者：朱春平[1,2] 李燕[1] 吴亚丽[1] 常婧竹[1] 刘琳[1] 高文明[1] 李新生[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]中国兽医药品监察所,北京100081

出　　处：《西北农林科技大学学报（自然科学版）》2015年第3期1-6,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家农业科技成果转化资金项目(2012GB2D000273);河南省科技成果转化计划项目(122201110027)

摘　　要：【目的】克隆和表达H3N2型猪流感病毒(SIV)A/swine/Henan/1/2010(H3N2)的核蛋白(NP)基因,为制备NP抗体和SIV基因工程疫苗奠定基础。【方法】提取SIV A/swine/Henan/1/2010(H3N2)的RNA,用RT-PCR扩增NP基因,将其定向克隆到pET-28a(+)原核表达载体,构建重组表达载体,并将其转化入大肠杆菌Rosetta中表达,用SDS-PAGE和Western blotting检测表达产物,同时考察IPTG不同浓度(0.2,0.4,0.6,0.8,1.0mmol/L)和不同诱导时间(2,4,6,8h)对重组NP蛋白表达量的影响。【结果】克隆到1 512bp的NP基因,与预期的核蛋白基因大小一致,共编码498个氨基酸;构建的重组表达载体在大肠杆菌Rosetta中表达出了分子质量约60ku的重组核蛋白,其主要以包涵体的形式存在。IPTG不同诱导浓度和不同诱导时间对重组NP蛋白表达量的影响均较小。【结论】克隆和表达了H3N2亚型猪流感病毒的核蛋白基因。【Objective】The nucleoprotein(NP)gene from swine influenza virus(SIV)A/swine/Henan/1/2010(H3N2)was cloned and expressed to lay foundation for the preparation of NP antibody and genetic engineering vaccine of SIV.【Method】The total RNA of SIV A/swine/Henan/1/2010(H3N2)was extracted.The NPgene was amplified from the total RNA by using RT-PCR and it was inserted into prokaryotic expression vector pET-28a(+)to construct recombinant expression vector.Then the vector was transformed and expressed in Escherichia coli Rosetta.The expressed protein was detected by SDS-PAGE and Western blotting,and the expression levels of recombinant NP protein under different IPTG concentrations(0.2,0.4,0.6,0.8,and 1.0 mmol/L)and different induction times(2,4,6,and 8h)were determined.【Result】The cloned NPgene(1 512bp)was consistent with the expected nucleoprotein gene,en-coding 498 amino acids.Recombinant expression vector expressed in E.coli Rosetta as recombinant nucleoprotein with the relative molecular weight of^60ku and mainly in the form of inclusion bodies.The expression level of recombinant NP protein was slightly influenced by IPTG concentration and induction time.【Conclusion】H3N2swine influenza virus nucleoprotein gene was successfully cloned and expressed.

关 键 词：猪流感病毒 核蛋白 H3N2亚型 大肠杆菌 

分 类 号：S852.65[农业科学—基础兽医学]