PCV2感染性分子克隆的制备及体内外感染性试验  

In vitro and in vivo infection experiment of infectious PCV2 molecular clone

作　　者：韩凌霞[1] 刘建华[2] 陈艳[2] 仇华吉[2] 王云峰[2] 童光志[2] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所实验动物中心,黑龙江哈尔滨150001 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2006年第3期241-247,270,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省科技攻关项目(编号:23010319621008)

摘　　要：将猪圆环病毒2型(Type2porcinecircovirus,PCV2)JXL株cDNA插入到载体pMD18-T中,获得的pMDT-PCV转染猪肾细胞PK15细胞系,盲传4代后,利用PCV2阳性猪血清进行间接免疫荧光抗体试验(IFA),能检测到特异性荧光。将pMDT-PCV质粒DNA经腹股沟淋巴结注射40日龄仔猪2头,分别接种2、3次,接种剂量500μg/次,首次注射35d心脏放血致死。在体内感染性试验期间,动物未出现明显临床症状。试验结束,病理切片结果显示,不同淋巴组织中分别存在淋巴细胞缺失或增多的现象;肾小球、扁桃体、空肠淋巴结和股前淋巴结等冰冻组织切片中存在大量PCV2病毒抗原;血清中PCV2特异性的ELISA抗体效价达12∶560,IFA效价为11∶280;通过聚合酶链式反应(PCR)可以分别从体外感染PK15细胞和体内感染动物及同圈饲养的空白对照猪的淋巴组织中扩增到病毒特异性基因片段。本研究证明含有单拷贝PCV2的重组质粒pMDT-PCV在体内、外均具有感染性,能衍生出病毒,并产生符合自然感染PCV2的大体病变和组织病理学变化,并激发较强的体液免疫应答。Infection of animals with a molecular viral clone is critical to study the genetic determinants of viral replication and virulence in the host. In this study, an infectious molecular DNA clone of porcine circovirus （PCV2） genome, named as pMDT-PCV, was constructed and used for infection experiments in vivo and in vitro, PK15 cell line was transfected with the DNA clone; and two 40-day-old piglets were inoculated through the superficial iliac lymph nodes with the dose of 500 μg/time for identification of its infectious ability, Histopathological lesions and PCV2-specific antigen were detected in several lymphoid tissues and major organs, including kidney, tonsil, lymph nodes spleen and heart by H,E, and immunohistological assay. The PCV2-specific antibody titers were 1：2 560 （ELISA） or 1：1 280 （IFA） in the serum of 35 day post inoculation. Two different particular fragments of PCV2 genome were amplified from DNA-inoculated animals and transfected-PK15 cultures, The results indicate that the cloned PCV2 genomic DNA is infectious in vivo and in vitro and the derived virus could replicate and induce a strong humoral immunity in pigs.

关 键 词：猪圆环病毒2 病毒分子克隆 感染性 抗体试验 

分 类 号：S852.659.2[农业科学—基础兽医学]