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。
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.
出处
《新疆农业大学学报》
CAS
北大核心
2010年第2期137-141,共5页
Journal of Xinjiang Agricultural University
基金
新疆维吾尔自治区高新技术项目(200611107)
关键词
犬瘟热病毒
N蛋白基因
克隆
原核表达
抗原性
canine distemper virus
CDV N protein gene
cloning
prokaryotic expression
antigenicity
