猪圆环病毒2型遗传标记毒株的构建及生物学特性鉴定

以临床分离的猪圆环病毒2型（PCV2）为材料，利用PCR法扩增病毒基因组，将2个基因组顺式连接插入到质粒载体中构建感染性分子克隆。通过引物设计替换碱基，在病毒基因组内插入Sal Ⅰ限制性内切酶位点作为遗传标记，经重组质粒转染细胞获得带有遗传标记的新毒株。采用免疫过氧化物酶单层细胞试验、免疫电镜、核酸序列分析表明，在病毒感染细胞中检出病毒特异抗原，其抗原性、病毒形态学及基因序列与亲本毒株一致。鉴于新病毒基因组内插入一个SalⅠ酶切住点，用PCR与限制性片段长度多态性分析法可与野生型病毒相鉴别。新毒株经细胞连续传60代，体外培养增殖性能稳定，毒价可达10^6.6CID50/mL。取病毒培养物经静脉和滴鼻途径接种35日龄抗体阴性仔猪4头，接种后表现出体温升高、进行性消瘦、被毛粗糙及体表淋巴结肿胀症状，迫杀后多种脏器中均能检测到病毒抗原与核酸。研究表明，利用感染性分子克隆手段构建带有遗传标记的PCV2新毒株，为进一步开展该病毒的致病性、疫苗免疫、分子诊断等研究奠定了基础。

Construction of genetic marker virus for porcine circovirus type 2 and identification of its biological characters

The complete genome of the PCV2 from field isolate was amplified by PCR.Two copies of the complete PCV2 genome were ligated in tandem into the plasmid vector to produce the PCV2 molecular DNA clone.The restriction enzyme Sal I site as genetic marker was inserted into the virus genome by substituting nucleotide in the primer designs.The infectivity of the PCV2 molecular DNA clone was determined by in vitro transfection of the cells.The antigenicity and morphology of the virus was confirmed by immunoperoxidase monolayer assay(IPMA)and immune electron microscope.The virus specific antigen was visualized by IPMA in the nucleus and cytoplasm of the infected cells.The viral genome was sequenced and could be differentiated with the wild type of PCV2 by PCR and restriction fragment length polymorphism(RFLP).The virus passed in the cell culture for sixty passages,and kept stable multiplication.The virus stock prepared from the sixtieth passage was determined to be 10~(6.6) TCID_(50)/mL.Four 35-day-old pigs of PCV2 antibody negative were inoculated with the virus by vein and nose routes. The pigs showed temperature high,progressive weight loss and lymph node swell symptom.The antigen and nucleic acid were detected in the numerous tissues and organs of the pigs.The results indicated that the cloned PCV2 genomic DNA may generate a genetic marker virus;the virus should be an useful tool for future studies of PCV2 pathogenesis,vaccination and molecular diagnosis.