H5N1亚型禽流感病毒HA蛋白的原核表达
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摘要
血凝素(hemagglutinin,HA)是禽流感病毒的主要抗原之一,原核表达HA是该抗原特性研究和疫苗制备的关键。利用分子生物学方法构建H5N1亚型的禽流感病毒HA基因原核表达载体,转化至大肠埃希菌Rosetta菌株,用不同IPTG浓度和时间诱导HA表达,检测蛋白表达量,分析不同诱导条件对HA表达效率的影响,以确定最佳诱导条件。重组表达载体pET32a-HA经BamHⅠ和EcoRⅠ双酶切鉴定,获得预期大小的DNA片段,说明HA基因成功插入质粒pET32a。Western blot检测结果表明,IPTG诱导重组菌株成功表达HA蛋白,当诱导浓度为0.1mmol/L,诱导时间为6h时,HA蛋白表达量最高。研究结果为HA抗原制备及禽流感疫苗的研究奠定基础。
Hemagglutinin(HA)is one of vital antigens of avian influenza virus.Expression of HA in prokaryotic cells plays an important role in exploration of HA characteristics and preparation of vaccine.In this study,HA of H5 N1 was cloned into the prokaryotic expression vector,and transformed into Escherichia coli Rosetta.In order to optimize induction conditions,recombinant bacteria were induced with different IPTG concentrations and induction time,and then ratio of target protein/total proteins was measured.The result of BamHⅠand EcoRⅠdigestion of pET32 a-HA demonstrated that the expected length of HA fragment was inserted into pET32 a.Western blot result illustrated that HA protein was successfully expressed in the recombinant strain via IPTG induction.With optimal conditon of 0.1 mmol/L IPTG and 6 h induction time,HA expression level reached highest.This study provided the foundation for the preparation of HA antigen and avian influenza vaccine research.
Hemagglutinin(HA)is one of vital antigens of avian influenza virus.Expression of HA in prokaryotic cells plays an important role in exploration of HA characteristics and preparation of vaccine.In this study,HA of H5 N1 was cloned into the prokaryotic expression vector,and transformed into Escherichia coli Rosetta.In order to optimize induction conditions,recombinant bacteria were induced with different IPTG concentrations and induction time,and then ratio of target protein/total proteins was measured.The result of BamHⅠand EcoRⅠdigestion of pET32 a-HA demonstrated that the expected length of HA fragment was inserted into pET32 a.Western blot result illustrated that HA protein was successfully expressed in the recombinant strain via IPTG induction.With optimal conditon of 0.1 mmol/L IPTG and 6 h induction time,HA expression level reached highest.This study provided the foundation for the preparation of HA antigen and avian influenza vaccine research.
引文
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[3]王念晨,曲楠楠,欧阳国文,等.当前我国H5亚型高致病性禽流感流行情况分析[J].中国动物传染病学报,2016,24(4):77-81.
[4]谭伟,谢丽基,谢芝勋,禽流感病毒疫苗研究进展[J].南方农业学报,2014,45(8):1492-1497.
[5]谭伟,徐倩,谢芝勋,禽流感病毒研究概述[J].基因组学与应用生物学,2014,33(1):194-199.
[6]王莎,唱韶红,刘波,等.H7N9流感病毒血凝素HA在毕赤酵母中的表达及免疫原性分析[J].军事医学,2015,39(6):448-452.
[7]纪方晓,陈济铛,石庆伟,等.H3N2亚型猪流感病毒HA和HA1蛋白的原核表达[J].动物医学进展,2016,37(6):22-25.
[8]成艳辉,黄维娟,李希妍,等.奥司他韦对我国A(H3N2)亚型流感病毒红细胞凝集和抑制试验的影响[J].病毒学报,2017,33(1):13-18.
[9]陶玲,李慧君,吴卫东.人感染H7N9亚型禽流感病毒分子生物学研究进展[J].新乡医学院学报,2017,34(9):779-785.
[10]陈欣,张乐萃,卢春晓,等.H9N2亚型禽流感病毒辽宁分离株HA1蛋白的原核表达[J].动物医学进展,2013,34(10):89-91.
[11]Linster M,Vanboheemen S,Degraaf M,et al.Identification,characterization,and natural selection of mutations driving airborne transmission of A/H5N1virus[J].Cell,2014,157(2):329-339.
[12]蔡若鹏,杨文涛,杨桂连,等.甲型流感病毒NP和M2的特性及其在疫苗研制中的应用[J].中国兽医学报,2014,34(10):1704-1708.
[13]谢辛慈,刘璐,袁松华,等.广谱流感疫苗的研究进展[J].微生物与感染,2016,11(1):48-54.
[14]Blanchfield K,Kamal R P,Tzeng W,et al.Recombinant influenza H7hemagglutinins induce lower neutralizing antibody titers in mice than do seasonal hemagglutinins[J].Influenza&Other Respiratory Viruses,2015,8(6):628-635.
[15]Cao W,Liepkalns J,Hassan A O,et al.A highly immunogenic vaccine against A(H7N9)influenza virus[J].Vaccine,2016,34(6):744-749.
[16]赵春丽,范秀军,赵冰,等.IPTG诱导浓度对重组人肝再生增强因子基因表达的影响[J].东北农业大学学报,2004(4):441-445.
[17]黄瑜,张雪利,鲁义善,等.红笛鲷tdt基因融合蛋白原核表达条件的优化及纯化[J].广东海洋大学学报,2013(1):44-49.
[18]庞耀珊,谢芝勋,谢丽基,等.H5N1亚型AIVHA抗原表位的高效表达及其抗原活性分析[J].基因组学与应用生物学,2012(2):109-116.