犬瘟热病毒N基因原核表达及多克隆抗体的制备
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摘要
采用PCR方法扩增犬瘟热病毒N基因,将其克隆至原核表达载体p ET-32a(+)中,构建犬瘟热N基因原核表达重组质粒,然后转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达重组N蛋白。从包涵体中纯化重组蛋白,制备多克隆抗体,采用Western blot检测其特异性。结果显示PCR扩增得到犬瘟热N基因,重组N蛋白在大肠杆菌BL21(DE3)菌株中得到表达,制备的多克隆抗体能与N蛋白特异性反应。
To study the function of N protein of Canine distemper virns(CDV), the CDV-N gene was amplified in PCR and cloned into expression vector p ET-32a(+). Subsequently, the recombinant plasmid p ET-32a-N was transformed into E.coli BL21(DE3) cells prior to induction with IPTG. The CDV-N protein was purified from the inclusion bodies. The PCR product of the CDV N gene was 1572 bp in length. The recombinant N protein was used to immunize rabbits to produce polyclonal antibodies. The rabbit antiserum was sampled and its specificity for N protein was confirmed in Western blot.
To study the function of N protein of Canine distemper virns(CDV), the CDV-N gene was amplified in PCR and cloned into expression vector p ET-32a(+). Subsequently, the recombinant plasmid p ET-32a-N was transformed into E.coli BL21(DE3) cells prior to induction with IPTG. The CDV-N protein was purified from the inclusion bodies. The PCR product of the CDV N gene was 1572 bp in length. The recombinant N protein was used to immunize rabbits to produce polyclonal antibodies. The rabbit antiserum was sampled and its specificity for N protein was confirmed in Western blot.
引文
[1]赵建军,柴秀丽,王凤雪,等.水貂犬瘟热CDV_3疫苗株基因组序列测定及H基因遗传稳定性分析[J].经济动物学报,2009,(1):13-20.
[2]郭玲,雷燕,陈世界,等.基于H蛋白基因的犬瘟热病毒遗传变异分析[J].畜牧兽医学报,2012,23(1):98-104.
[3]Demeter Z,Lakatos B,Palade E A,et al.Genetic diversity of Hungarian canine distemper virus strains[J].Vet Microbiol,2007,122(3-4):258-269.
[4]朱春生,赵建军,白雪,等.水貂狐狸及貉源犬瘟热病毒H基因的克隆与序列分析[J].中国兽医科学,2014,44(2):134-139.
[5]Li W,Li T,Liu Y,et al.Genetic characterization of an isolate of canine distemper virus from a Tibetan Mastiff in China[J].Virus Genes,2014,491:109-112.
[6]Mochizuki M,Hashimoto M,Hagiwara S,et al.Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan[J].J Clin Microbiol,1999,37(9):2936-2942.
[7]Calderon M G,Remorini P,Periolo O,et al.Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina[J].Vet Microbiol,2007,125(3-4):341-349.
[8]金艺鹏,刘巧荣,孙明,等.大熊猫源犬瘟热病毒基因组遗传特征分析[J].中国农业科学,2015,(7):1445-1452.
[9]王君玮.貂、狐、貉源犬瘟热病毒分离鉴定与分子生物学特性研究[D].南京:南京农业大学,2008.
[10]Bolt G,Jensen T D,Gottschalck E,et al.Genetic diversity of the attachment(H)protein gene of current field isolates of canine distemper virus[J].J Gen Virol,1997,78(Pt2):367-372.
[11]程悦宁.犬瘟热病毒SY-12株的分离鉴定及其全基因组序列测定与分析[D].长春:吉林农业大学,2014.
[12]Stern L B,Greenberg M,Gershoni J M,et al.The hemagglutinin envelope protein of canine distemper virus(CDV)confers cell tropism as illustrated by CDV and measles virus complementation analysis[J].J Virol,1995,69(3):1661-1668.
[13]Sultan S,Charoenvisal N,Lan N T,et al.The Asia 2specific signal peptide region and other domains in fusion protein genes characterized Asia 1 and Asia 2canine distemper viruses[J].Virol J,2009,6:157.
[14]宗春苗.水貂犬源犬瘟热病毒的分离鉴定及H基因的序列分析[D].长春:吉林农业大学,2007.
[2]郭玲,雷燕,陈世界,等.基于H蛋白基因的犬瘟热病毒遗传变异分析[J].畜牧兽医学报,2012,23(1):98-104.
[3]Demeter Z,Lakatos B,Palade E A,et al.Genetic diversity of Hungarian canine distemper virus strains[J].Vet Microbiol,2007,122(3-4):258-269.
[4]朱春生,赵建军,白雪,等.水貂狐狸及貉源犬瘟热病毒H基因的克隆与序列分析[J].中国兽医科学,2014,44(2):134-139.
[5]Li W,Li T,Liu Y,et al.Genetic characterization of an isolate of canine distemper virus from a Tibetan Mastiff in China[J].Virus Genes,2014,491:109-112.
[6]Mochizuki M,Hashimoto M,Hagiwara S,et al.Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan[J].J Clin Microbiol,1999,37(9):2936-2942.
[7]Calderon M G,Remorini P,Periolo O,et al.Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina[J].Vet Microbiol,2007,125(3-4):341-349.
[8]金艺鹏,刘巧荣,孙明,等.大熊猫源犬瘟热病毒基因组遗传特征分析[J].中国农业科学,2015,(7):1445-1452.
[9]王君玮.貂、狐、貉源犬瘟热病毒分离鉴定与分子生物学特性研究[D].南京:南京农业大学,2008.
[10]Bolt G,Jensen T D,Gottschalck E,et al.Genetic diversity of the attachment(H)protein gene of current field isolates of canine distemper virus[J].J Gen Virol,1997,78(Pt2):367-372.
[11]程悦宁.犬瘟热病毒SY-12株的分离鉴定及其全基因组序列测定与分析[D].长春:吉林农业大学,2014.
[12]Stern L B,Greenberg M,Gershoni J M,et al.The hemagglutinin envelope protein of canine distemper virus(CDV)confers cell tropism as illustrated by CDV and measles virus complementation analysis[J].J Virol,1995,69(3):1661-1668.
[13]Sultan S,Charoenvisal N,Lan N T,et al.The Asia 2specific signal peptide region and other domains in fusion protein genes characterized Asia 1 and Asia 2canine distemper viruses[J].Virol J,2009,6:157.
[14]宗春苗.水貂犬源犬瘟热病毒的分离鉴定及H基因的序列分析[D].长春:吉林农业大学,2007.