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犬瘟热病毒N蛋白基因的克隆、原核表达及其表达产物抗原性鉴定
引用本文:申卫红,马素贞,陈胜男,刘腾飞,陶玉成,简子健.犬瘟热病毒N蛋白基因的克隆、原核表达及其表达产物抗原性鉴定[J].新疆农业大学学报,2010,33(2):137-141.
作者姓名:申卫红  马素贞  陈胜男  刘腾飞  陶玉成  简子健
作者单位:新疆农业大学动物医学学院,乌鲁木齐,830052
基金项目:新疆维吾尔自治区高新技术项目 
摘    要:参考GenBank中发表的CDVN蛋白基因序列,用Oligo6.0软件设计一对特异性引物,从患犬瘟热的犬全血样品中提取总RNA,经RT—PCR扩增得到1572bp的基因片段,将其插入pMD18-T克隆载体中构建了pMD18-CDVN重组质粒,将其转入到大肠杆菌DH5a中,进行鉴定、测序。将目的基因与原核表达载体pGEX-4T-2连接构建了pGEX-4T-2-CDVN重组质粒,转化到大肠杆菌BL21(DE3)中,进行鉴定、测序、IPTG诱导表达。序列分析表明克隆的CDVN蛋白基因从起始密码子ATG开始到终止密码子TAA结束全长为1572个核苷酸,与GenBank中发表的CDVN蛋白基因序列相比同源率达到93.9%~99.1%。Western-blotting分析显示表达产物为84kDa的融合蛋白,可被犬瘟热抗血清所识别,具有良好的抗原性。

关 键 词:犬瘟热病毒  N蛋白基因  克隆  原核表达  抗原性

Cloning and Prokaryotic Expression of N Protein Gene in Canine Distemper Virus(CDV)and Identification of Antigenicity of Expressive Products
SHEN Wei-hong,MA Su-zhen,CHEN Sheng-nan,LIU Teng-fei,TAO Yu-cheng,JIAN Zi-jian.Cloning and Prokaryotic Expression of N Protein Gene in Canine Distemper Virus(CDV)and Identification of Antigenicity of Expressive Products[J].Journal of Xinjiang Agricultural University,2010,33(2):137-141.
Authors:SHEN Wei-hong  MA Su-zhen  CHEN Sheng-nan  LIU Teng-fei  TAO Yu-cheng  JIAN Zi-jian
Institution:SHEN Wei-hong,MA Su-zhen,CHEN Sheng-nan,LIU Teng-fei,TAO Yu-cheng,JIAN Zi-jian(College of Animal Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
Abstract:One pair of the special primers were designed by Oligo6.0 software according to the nucleocapsid (N) protein gene sequences of canine distemper virus (CDV) published in GenBank. A 1 572 bp CDV N protein gene fragment was amplified by RT-PCR from the total RNA template in the whole blood samples infected with CDV and was cloned into a pMD18-T vector in order to construct pMD18-CDV N recombination plasmid and then transformed into the E. coli DH5α before sequencing and identification. The cloned N protein gene from CDV was subcloned into expression vector pGEX-4T-2 and the resultant recombinant plasmid was transformed into E. coli BL21(DE3) cells for sequencing, identifying and expressed by IPTG Induced. Sequence analysis was shown that the total length of CDV N protein gene was 1572 nucleotides from start codon ATG to stop codon TAA, the homology of the nucleotide sequence was 93.9%-99.1% compared with published in GenBank. Western-blotting analysis was shown that a fusion protein of 84 kDa was expressed which could be recognized by canine antiserum. This result was indicated the fusion protein (GST-CDV N) possessed strong antigenicity which could be used as a recombinant antigen to develop a subunit vaccine.
Keywords:canine distemper virus  CDV N protein gene  cloning  prokaryotic expression  antigenicity  
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