犬细小病毒VP2基因的扩增、序列分析及其原核表达  

Amplification,sequence analysis and prokaryotic expression of canine parvovirus VP2 gene

作　　者：王园园[1] 张馨颖 陈漫[3] 董晓琳[3] 孙钰[3] 孙瑞[3] 王习文[3] 李志萍[3] 曲晗[3] 焦虎平[1] 靳朝[1] 夏志平[3] 

机构地区：[1]吉林大学动物医学学院,长春130062 [2]吉林农业大学,长春130118 [3]中国人民解放军军事医学科学院军事兽医研究所基础兽医研究室,长春130122

出　　处：《黑龙江畜牧兽医》2016年第9期37-41,288,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家科技基础条件平台建设计划项目(2011ZXJ09201-031)

摘　　要：为了制备犬细小病毒（CPV）VP2蛋白抗原，试验采用PCR方法，以一例疑似CPV感染的出血性肠炎患犬粪便为样品对VP2基因进行扩增，并将该目的基因克隆到pMD18-T质粒上。进行EcoRI／HindⅢ双酶切鉴定及测序鉴定；对该基因进行序列分析后将VP2基因克隆到原核表达载体pET-32a上，并转化E．coliBL21（DE3），诱导表达后进行Ni-NTA亲和层析纯化、Western—blot试验验证其生物活性。结果表明：克隆的基因全长为1790bp；该基因序列与已报道的CPV基因序列同源性高达99．2％～99．9％，氨基酸同源性达99．0％～99．8％，为新CPV-2a基因亚型，其中与泰国株GQ379042．1亲缘性最高，仅1个碱基不同，与猫细小病毒同源性高达98．69％；获得了87ku的目的蛋白。说明该临床病例确为CPV感染。该株细小病毒VP2蛋白的成功表达可为吉林省CPV流行病学调查提供资料，可用于进一步的疫苗研究及免疫学方法的建立。To prepare canine parvovirus （CPV）VP2 protein antigen, the VP2 gene was amplified by PCR taking the hemorrhagic enteritis feces from a ease of suspected CPV infection as the samples. The target gene was cloned into a plasmid pMD18 -T, and then the recombinant plas- mid was identified by double restriction enzyme digestion with EeoRl/HindllI and nucleotide sequencing; after analyzing this gene, the VP2 gene was cloned into a prokaryotie expression vector pET -32a and transformed into E. coli BL21 （ DE3 ） , and then its biological activity was verified by purification using Ni - NTA affinity chromatography and Western - blot test after induction of expression. The results showed that the full length of cloned gene was 1709 bp. The VP2 gene sequences shared 99.2 % to 99.9 % with the reported gene sequences of CPV, and the amino acid sequence homology Was up to 99. 0 % to 99.8 %. It was confirmed that the CPV - ZXY strain was a new CPV -2a subtype, there- into, the CPV - ZXY strain was highly homologous to Thailand strain GQ379042.1 by only a different base, and shared 98.69 % homology to feline paravirus （FPV）. The target protein Of 87 KD was Obtained. The results indicate that the clinical case is indeed CPV infection. Success- ful expression of the VP2 protein of CPV can provide a material for the epidemiological investigation of CPV in Jilin province , which can be used for the further vaccine research and the establishment of immunological methods.

关 键 词：犬细小病毒(CPV) 新CPV-2a亚型 同源性分析 遗传进化树 原核表达 

分 类 号：S852.659.2[农业科学—基础兽医学]