H6N6亚型禽流感病毒N6基因的原核表达与多克隆抗体制备
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摘要
为表达H6N6亚型禽流感病毒(avian influenza virus,AIV)神经氨酸酶(NA)蛋白,并制备多克隆抗体,本试验根据GenBank中AIV N6基因序列(登录号:MG434500)设计特异性引物,对贵州地区分离的H6N6亚型AIV贵州株进行N6基因PCR扩增,将其克隆到原核表达载体pET-32a(+)中,构建重组原核表达载体pET-32a-N6,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达His-N6重组蛋白,将诱导产物超声破碎离心后,利用镍柱对重组蛋白进行纯化,并利用SDS-PAGE和Western blotting对表达蛋白进行双重鉴定,将纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体,并通过间接ELISA检测其效价。结果显示,AIV贵州株N6基因编码区长1 380bp,可编码459个氨基酸,其中175-207bp缺失33个核苷酸;重组质粒pET-32a-N6经双酶切鉴定分别获得大小为5 900bp左右的载体条带和1 380bp左右的目的基因条带,成功构建了pET-32a-N6重组质粒;蛋白超声破碎后经SDS-PAGE发现,重组蛋白主要存在于沉淀中,以包涵体形式存在,蛋白分子质量约为70ku,与预期结果一致;纯化后的重组蛋白经SDS-PAGE和Western blotting双重鉴定,均在70ku处出现条带,说明纯化的蛋白为重组蛋白pET-32a-N6,表达产物具有免疫学活性,包涵体经变性、复性处理,重组表达蛋白分别被His抗体和兔源抗N6多克隆抗体所识别。间接ELISA检测其效价高于1∶3 200。以上结果表明,试验成功克隆、表达了H6N6亚型AIV的N6基因,所制备的N6蛋白多克隆抗体具有良好的免疫活性,能被His抗体和兔源抗N6多克隆抗体所识别。
This experiment was aimed to express recombinant neuraminidase(NA)protein of H6 N6 subtype avian influenza virus(AIV)isolated from Guizhou province,and prepared polyclonal antibody against NA protein of AIV.The specific primers were designed according to N6 gene of AIV in GenBank(accession No.:MG434500),and used for amplification of N6 gene by PCR.PCR product was cloned into the expression vector pET-32 a(+).The soluble recombinantprotein was expressed in E.coli BL21(DE3)with IPTG.The expression product was sonicated and purified by nickel column.The recombinant proteins were identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared from the rabbit immunized with purified recombinant protein.The titer of the antibody was detected by indirect ELISA.The results showed that coding region of N6 gene was 1 380 bp,which encoded 459 amino acids.There were 33 nucleotides deletion from position 175 to 207 bp at N6 gene stalk.The recombinant plasmid pET-32 aN6 was identified by double enzyme digestion,it appeared about 5 900 bp vector band and about1 380 bp target band,pET-32 a-N6 recombinant plasmid was successfully constructed;SDS-PAGE analysis result showed the molecular weight of the recombinant N6 protein was about 70 ku which consistented with expected result.It showed that the target gene was successfully expressed in the form of inclusion body.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and showed a band of 70 ku in size,indicating that the purified protein was the recombinant protein pET-32 a-N6.The purified protein of denaturation and renaturation could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum,respectively.The titer of the antibody was about 1:3 200 by detection of indirect ELISA.All the results indicated that N6 gene of H6N6 subtype AIV was successfully cloned and expressed,the prepared anti-N6 showed good immune activity,and the protein purified could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum.
This experiment was aimed to express recombinant neuraminidase(NA)protein of H6 N6 subtype avian influenza virus(AIV)isolated from Guizhou province,and prepared polyclonal antibody against NA protein of AIV.The specific primers were designed according to N6 gene of AIV in GenBank(accession No.:MG434500),and used for amplification of N6 gene by PCR.PCR product was cloned into the expression vector pET-32 a(+).The soluble recombinantprotein was expressed in E.coli BL21(DE3)with IPTG.The expression product was sonicated and purified by nickel column.The recombinant proteins were identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared from the rabbit immunized with purified recombinant protein.The titer of the antibody was detected by indirect ELISA.The results showed that coding region of N6 gene was 1 380 bp,which encoded 459 amino acids.There were 33 nucleotides deletion from position 175 to 207 bp at N6 gene stalk.The recombinant plasmid pET-32 aN6 was identified by double enzyme digestion,it appeared about 5 900 bp vector band and about1 380 bp target band,pET-32 a-N6 recombinant plasmid was successfully constructed;SDS-PAGE analysis result showed the molecular weight of the recombinant N6 protein was about 70 ku which consistented with expected result.It showed that the target gene was successfully expressed in the form of inclusion body.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and showed a band of 70 ku in size,indicating that the purified protein was the recombinant protein pET-32 a-N6.The purified protein of denaturation and renaturation could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum,respectively.The titer of the antibody was about 1:3 200 by detection of indirect ELISA.All the results indicated that N6 gene of H6N6 subtype AIV was successfully cloned and expressed,the prepared anti-N6 showed good immune activity,and the protein purified could be specifically recognized by His-antibody and rabbit-anti-N6 hyperimmune serum.
引文
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[8]柴洪亮.禽流感病毒分离株A/Mallard/ZhaLong/88/04(H4N6)全基因的克隆及序列分析[D].哈尔滨:东北林业大学,2006.CHAI H L.Molecular analysis of the entire genes of an avian influenza virus isolate A/Mallard/ZhaLong/88/04(H4N6)[D].Harbin:Northeast Forestry University,2006.(in Chinese)
[9]谭刘刚,鲁梅,朱凤株,等.1株中国北方鸭H4N6亚型禽流感病毒的分子特征[J].中国兽医学报,2015,35(2):207-212.TAN L G,LU M,ZHU F Z,et al.Characterization of an avian influenza virus of subtype H4N6isolated from ducks in Northern China[J].Chinese Journal Veterinary Science,2015,35(2):207-212.(in Chinese)
[10]解林红,高晓龙,耿海东,等.H4N6亚型禽流感病毒分离与氨基酸序列测定[J].中国病原生物学杂志,2015,10(5):385-388.XIE L H,GAO X L,GENG H D,et al.Isolation and amino acid sequencing of avian influenza virus subtype H4N6[J].Journal of Pathogen Biology,2015,10(5):385-388.(in Chinese)
[11]万春和,傅光华,程龙飞,等.番鸭源H6N6亚型禽流感病毒全基因组的分子特征[J].微生物学报,2010,50(9):1147-1154.WAN C H,FU G H,CHENG L F,et al.Molecular characterization analysis of a muscovy duck-origin H6N6subtype avian influenza virus[J].Acta Microbiologica Sinica,2010,50(9):1147-1154.(in Chinese)
[12]陈佳琪,嵇辛勤,段志强,等.1株H6N6亚型禽流感病毒贵州分离株HA和NA基因克隆与序列分析[J].中国畜牧兽医,2016,43(2):296-303.CHEN J Q,JI X Q,DUAN Z Q,et al.Cloning and sequence analysis of HAand NAgenes of one strain of H6N6subtype avian influenza virus isolated from Guizhou province[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):296-303.(in Chinese)
[13]顾敏,宋庆庆,李彦芳,等.1株H6N6亚型禽流感病毒A/duck/Jiangsu/022/2009的全基因测序及遗传进化分析[J].中国人兽共患病学报,2010,26(7):642-647.GU M,SONG Q Q,LI Y F,et al.Genome sequencing and genetic analysis of one H6N5subtype avian influenza virus A/duck/Jiangsu/022/2009[J].Chinese Journal of Zoonoses,2010,26(7):642-647.(in Chinese)
[14]祁思敏,宋勇春,崔仑标,等.1株H6N6亚型禽流感病毒分子遗传特性分析[J].微生物与感染,2017,12(5):287-293.QI S M,SONG Y C,CUI L B,et al.Molecular characterization of one H6N6 subtype avian influenza virus[J].Journal of Microbes and Infection,2017,12(5):287-293.(in Chinese)
[15]葛志闯,孙文强,刘东,等.一株鸭源H6N6亚型禽流感病毒的基因组测序及遗传进化分析[J].中国家禽,2017,39(14):21-28.GE Z C,SUN W Q,LIU D,et al.Genome sequencing and genetic evolutionary analysis of a duck-origin H6N6subtype avian influenza virus[J].China Poultry,2017,39(14):21-28.(in Chinese)
[16]高晓龙,范胜涛,李胜楠,等.中国首株H13N6亚型禽流感病毒遗传进化分析及致病性和传播性评估[J].中国预防兽医学报,2015,37(3):172-176.GAO X L,FAN S T,LI S N,et al.Phylogenetic analysis and assessment of the pathogenicity and transmissibility of the H13N6subtype AIV firstly isolated from China[J].Chinese Journal Preventive Veterinary Medicine,2015,37(3):172-176.(in Chinese)
[17]熊文婕,谢芝勋,李孟,等.一株鸭源H1N6亚型禽流感病毒的分离鉴定及HA和NA基因序列分析[J].中国畜牧兽医,2018,45(2):310-319.XIONG W J,XIE Z X,LI M,et al.Isolation,identification and genetic analysis of HA[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):310-319.(in Chinese)
[18]HERRMANN B,LARSSON C,ZWEYGBERG B W.Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR:Comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay(FLUOIA)[J].Journal of Clinical Microbiology,2001,39(1):134-138.
[19]QIU B F,LIU W,PENG D,et al.A reverse transcription PCR for subtyping of the neuraminidase of avian influenza viruses[J].Journal of Viological Methods,2009,15(5):193-198.
[20]王利,李康生.禽流感病毒的免疫原性及其疫苗的研究进展[J].微生物学免疫学进展,2006,16(2):54-57.WANG L,LI K S.Immunogenicity of avian influenza virus and research progress of in vaccine[J].Progress in Microbiology and Immunology,2006,16(2):54-57.(in Chinese)
[21]刘岩.1998~2002年间河南省H9N2亚型AIV的抗原性漂变研究[D].郑州:河南农业大学,2003.LIU Y.Antigenic drift of H9N2subtype avian influenza viruses in Henan province during 1998-2002[D].Zhengzhou:Henan Agricultural University,2003.(in Chinese)
[22]HAUSMANN J,KRETZSCHMAR E,GAREN W,et al.Biosynthesis,intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase role of unpaired cysteines and indivual oligosaccharides[J].Journal of General Virology,1997,78(12):3233-3245.
[23]SAIN T,KAWAOKA Y,WEBSTER R G.Phylogenetic analysis of the N8neuraminidase gene of influenza A viruses[J].Virology,1993,193(2):868-876.
[24]GARMAN E,LAVER G.Controlling influenza by inhibiting the virus’s neuraminidase[J].Current Drug Targets,2004,5(2):119-136.
[25]吴艳菊,张治业,倪小舒,等.H5N1亚型禽流感病毒NA和NS1蛋白原核表达及多克隆抗体制备[J].黑龙江畜牧兽医,2017,6:1-4.WU Y J,ZHANG Z Y,NI X S,et al.Prokaryotic expression of the NAand NS1genes of H5N1subtype aivian influenza virus and preparation of their polyclonal antibodies[J].Heilongjiang Animal Science and Veterinary Medicine,2017,6:1-4.(in Chinese)
[26]李清州,郝惠芳,王丽,等.H5N1亚型禽流感病毒血凝素(HA)基因的表达及纯化[J].河南农业科学,2008,12(8):127-130.LI Q Z,HAO H F,WANG L,et al.Expression and purification of the HAgene of the avian influenza virus subtype H5N1[J].Journal of Henan Agricultural Sciences,2008,12(8):127-130.(in Chinese)
[2]WEBSTER R G,BEANW J,GORMAN O T,et al.Evolution and ecology of influenza A viruses[J].Microbiology Review,1992,56(1):152-179.
[3]PUSHKO P,TRETYAKOVA I,HIDAJAT R,et al.Virus-like particles displaying H5,H7,H9hemagglutinins and N1neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens[J].Virology,2017,501(9):176-182.
[4]LI Q,SUN X,LI Z,et al.Structural and functional characterization of neuraminidase-like molecule N10derived from bat influenza A virus[J].Proceedings of the National Academy Sciences of the United States of America,2012,109(46):18897-18902.
[5]MATROSOVICH M N,MATROSOVICH T Y,GRAY T,et al.Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium[J].Virology,2004,78(22):12665-12667.
[6]BI Y H,CHEN Q C,WANG Q L,et al.Genesis,evolution and prevalence of H5N6avian influenza viruses in China[J].Cell Host&Microbe,2016,20(6):810-821.
[7]刘映霞,史卫峰,刘翟,等.H5N6禽流感病毒的基因起源、进化和流行[J].科学新闻,2017,6(4):104.LIU Y X,SHI W F,LIU Z,et al.Genesis,evolution and prevalence of H5N6avian influenza viruses in China[J].Science News,2017,6(4):104.(in Chinese)
[8]柴洪亮.禽流感病毒分离株A/Mallard/ZhaLong/88/04(H4N6)全基因的克隆及序列分析[D].哈尔滨:东北林业大学,2006.CHAI H L.Molecular analysis of the entire genes of an avian influenza virus isolate A/Mallard/ZhaLong/88/04(H4N6)[D].Harbin:Northeast Forestry University,2006.(in Chinese)
[9]谭刘刚,鲁梅,朱凤株,等.1株中国北方鸭H4N6亚型禽流感病毒的分子特征[J].中国兽医学报,2015,35(2):207-212.TAN L G,LU M,ZHU F Z,et al.Characterization of an avian influenza virus of subtype H4N6isolated from ducks in Northern China[J].Chinese Journal Veterinary Science,2015,35(2):207-212.(in Chinese)
[10]解林红,高晓龙,耿海东,等.H4N6亚型禽流感病毒分离与氨基酸序列测定[J].中国病原生物学杂志,2015,10(5):385-388.XIE L H,GAO X L,GENG H D,et al.Isolation and amino acid sequencing of avian influenza virus subtype H4N6[J].Journal of Pathogen Biology,2015,10(5):385-388.(in Chinese)
[11]万春和,傅光华,程龙飞,等.番鸭源H6N6亚型禽流感病毒全基因组的分子特征[J].微生物学报,2010,50(9):1147-1154.WAN C H,FU G H,CHENG L F,et al.Molecular characterization analysis of a muscovy duck-origin H6N6subtype avian influenza virus[J].Acta Microbiologica Sinica,2010,50(9):1147-1154.(in Chinese)
[12]陈佳琪,嵇辛勤,段志强,等.1株H6N6亚型禽流感病毒贵州分离株HA和NA基因克隆与序列分析[J].中国畜牧兽医,2016,43(2):296-303.CHEN J Q,JI X Q,DUAN Z Q,et al.Cloning and sequence analysis of HAand NAgenes of one strain of H6N6subtype avian influenza virus isolated from Guizhou province[J].China Animal Husbandry&Veterinary Medicine,2016,43(2):296-303.(in Chinese)
[13]顾敏,宋庆庆,李彦芳,等.1株H6N6亚型禽流感病毒A/duck/Jiangsu/022/2009的全基因测序及遗传进化分析[J].中国人兽共患病学报,2010,26(7):642-647.GU M,SONG Q Q,LI Y F,et al.Genome sequencing and genetic analysis of one H6N5subtype avian influenza virus A/duck/Jiangsu/022/2009[J].Chinese Journal of Zoonoses,2010,26(7):642-647.(in Chinese)
[14]祁思敏,宋勇春,崔仑标,等.1株H6N6亚型禽流感病毒分子遗传特性分析[J].微生物与感染,2017,12(5):287-293.QI S M,SONG Y C,CUI L B,et al.Molecular characterization of one H6N6 subtype avian influenza virus[J].Journal of Microbes and Infection,2017,12(5):287-293.(in Chinese)
[15]葛志闯,孙文强,刘东,等.一株鸭源H6N6亚型禽流感病毒的基因组测序及遗传进化分析[J].中国家禽,2017,39(14):21-28.GE Z C,SUN W Q,LIU D,et al.Genome sequencing and genetic evolutionary analysis of a duck-origin H6N6subtype avian influenza virus[J].China Poultry,2017,39(14):21-28.(in Chinese)
[16]高晓龙,范胜涛,李胜楠,等.中国首株H13N6亚型禽流感病毒遗传进化分析及致病性和传播性评估[J].中国预防兽医学报,2015,37(3):172-176.GAO X L,FAN S T,LI S N,et al.Phylogenetic analysis and assessment of the pathogenicity and transmissibility of the H13N6subtype AIV firstly isolated from China[J].Chinese Journal Preventive Veterinary Medicine,2015,37(3):172-176.(in Chinese)
[17]熊文婕,谢芝勋,李孟,等.一株鸭源H1N6亚型禽流感病毒的分离鉴定及HA和NA基因序列分析[J].中国畜牧兽医,2018,45(2):310-319.XIONG W J,XIE Z X,LI M,et al.Isolation,identification and genetic analysis of HA[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):310-319.(in Chinese)
[18]HERRMANN B,LARSSON C,ZWEYGBERG B W.Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR:Comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay(FLUOIA)[J].Journal of Clinical Microbiology,2001,39(1):134-138.
[19]QIU B F,LIU W,PENG D,et al.A reverse transcription PCR for subtyping of the neuraminidase of avian influenza viruses[J].Journal of Viological Methods,2009,15(5):193-198.
[20]王利,李康生.禽流感病毒的免疫原性及其疫苗的研究进展[J].微生物学免疫学进展,2006,16(2):54-57.WANG L,LI K S.Immunogenicity of avian influenza virus and research progress of in vaccine[J].Progress in Microbiology and Immunology,2006,16(2):54-57.(in Chinese)
[21]刘岩.1998~2002年间河南省H9N2亚型AIV的抗原性漂变研究[D].郑州:河南农业大学,2003.LIU Y.Antigenic drift of H9N2subtype avian influenza viruses in Henan province during 1998-2002[D].Zhengzhou:Henan Agricultural University,2003.(in Chinese)
[22]HAUSMANN J,KRETZSCHMAR E,GAREN W,et al.Biosynthesis,intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase role of unpaired cysteines and indivual oligosaccharides[J].Journal of General Virology,1997,78(12):3233-3245.
[23]SAIN T,KAWAOKA Y,WEBSTER R G.Phylogenetic analysis of the N8neuraminidase gene of influenza A viruses[J].Virology,1993,193(2):868-876.
[24]GARMAN E,LAVER G.Controlling influenza by inhibiting the virus’s neuraminidase[J].Current Drug Targets,2004,5(2):119-136.
[25]吴艳菊,张治业,倪小舒,等.H5N1亚型禽流感病毒NA和NS1蛋白原核表达及多克隆抗体制备[J].黑龙江畜牧兽医,2017,6:1-4.WU Y J,ZHANG Z Y,NI X S,et al.Prokaryotic expression of the NAand NS1genes of H5N1subtype aivian influenza virus and preparation of their polyclonal antibodies[J].Heilongjiang Animal Science and Veterinary Medicine,2017,6:1-4.(in Chinese)
[26]李清州,郝惠芳,王丽,等.H5N1亚型禽流感病毒血凝素(HA)基因的表达及纯化[J].河南农业科学,2008,12(8):127-130.LI Q Z,HAO H F,WANG L,et al.Expression and purification of the HAgene of the avian influenza virus subtype H5N1[J].Journal of Henan Agricultural Sciences,2008,12(8):127-130.(in Chinese)