摘要
从临床发病犬采集粪便样品,以F81传代细胞进行病毒分离,经血球凝集实验(HA)和血球凝集抑制实验(HI)初步鉴定为犬细小病毒。为进一步确诊,根据Genbank中已发表的犬细小病毒VP2基因序列设计并合成一对引物,通过聚合酶链反应(PCR)扩增出CPV VP2基因,酶切,测序加以鉴定。其序列与国际已发表的CPV-d(type 2)、CPV-1(5 type 2a)、CPV-3(9 type 2b)VP2序列同源性分别为98.97%,98.75%,98.69%,氨基酸序列的同源性分别为98.12%,97.60%,97.60%,从而证明此分离株为犬细小病毒。在获得VP2基因的基础上,为实现VP2蛋白的表达,构建了真核表达质粒pMel BacC-VP2。
The HL-01 isolate of canine parvovirus was propagated agglutination test （HA） and haem agglutination inhibition test （HI） on F81 cell. And identified by the haem Using a pair of primers based on the published sequence of CPV＇s VP2 gene, the full length gene encoding outer capsid protein VP2 was amplified from cell culture by polymerase chain reaction （PCR）. The VP2 gene of HL-01 isolate of canine parvovirus was inserted into pMD18-T vector and was identified by sequencing, digestion： The result showed that there was a high homology innucleotide sequence and aminoacid sequence incomparison with CPV-d （type 2）, CPV-15（type 2a） and CPV-39 （type 2b）. The rate of nucleotide sequence homology was 98.97%, 98.75%, 98.69%, and the aminoacid homology was 98.12%, 97.60%, 97.60%. The VP2 gene of the isolate was inserted into the eukaryotic expression plasmid pMel BacC and construced recombinated plasmid pMel BacC-VP2 in order to realize the expression of VP2 protein.
出处
《东北农业大学学报》
CAS
CSCD
2006年第1期69-73,共5页
Journal of Northeast Agricultural University
基金
黑龙江省科学技术计划项目(SY02-09)
关键词
犬细小病
VP2基因
克隆
表达质粒
构建
canine parvovirus
VP2
cloning
pMel BacC expression plasmid
construction
