非典型犬瘟热病毒核衣壳蛋白基因序列分析及其在大肠杆菌中的表达

Analysis and expression of nucleocapsid protein gene of atypical canine distemper virus in Escherichia coli

为分析当地非典型犬瘟热病毒(CDV)核衣壳蛋白(N)基因的序列特征及其表达产物的抗原性,根据已发表CDV的N基因序列设计引物,用RT-PCR方法从引起非典型症状的CDV细胞培养物中扩增N基因,进行克隆和序列分析,结果表明:该非典型CDV的N基因与已发表的12个CDV强毒株的核苷酸序列和氨基酸序列同源性分别在96.6%～99.2%和97.9%～99.4%之间,与已发表的4个CDV疫苗弱毒株的同源性分别在93.2%～93.6%和96.4%～97.5%之间;在N基因系统发育进化树上,非典型CDV与12个强毒株处在同一亚群,而且与9个中国分离毒株的亲缘关系近于3个国外毒株。N基因在大肠杆菌中表达的重组N蛋白的分子量为62 ku,主要以包涵体的形式存在;用western blot分析,重组N蛋白可与CDV阳性血清发生特异性反应;以纯化的重组N蛋白为抗原建立的CDV抗体间接ELISA检测方法具有良好的特异性。

To investigate the canine distemper virus （CDV） isolated from the lung and liver samples of an atypical clinical cases of canine distemper, the nucleocapsid protein （N） gene was amplified by RT-PCR with a pair of primers designed based on the N gene sequence of CDV reference strains in GenBank. The sequence analysis demonstrated that the homology of the atypical CDV N gene with other 12 virulent reference strains in GenBank were from 96.6% to 99.2% in nucleotide and 97.9% to 99.4% in amino acid sequence, and shared 93.2% to 93.6% nucleotide sequence homology and 96.4% to 97.5% amino acid sequence with that of 4 attenuated vaccine stains. The phylogenetic tree based on the sequences of the N gene showed that the atypical CDV was in the same subgroup with the virulent strains. Furthermore, the N gene from the atypical CDV was sub-cloned into pET-28a（＋） and expressed in E. coll. SDS-PAGE analysis indicated that the recombinant N protein was 62 ku and mainly existed as inclusion bodies in E. coll. Western blot showed that the recombinant N protein could be recognized by CDV positive serum. An indirectELISA coated with the purified recombinant N protein showed specificity for the detection of antibody against CDV.