论文部分内容阅读
设计带BamHⅠ酶切标记位点的引物,PCR扩增鹅圆环病毒(Goose circovirus,GoCV)全长基因组,将2个基因组顺式连接插入到pGEM-T Easy载体中,获得GoCV全长基因组头尾串联二聚体感染性克隆质粒pGEMT-2GoCV。EcoRⅠ酶切线性化pGEMT-2GoCV,与脂质体混合转染GoCV阴性鹅胚和雏鹅,常规PCR检测发现GoCV在转染鹅体内增殖,鹅胚转染组于孵出第2周和第4周检出血清阳性,且其中一个个体于4周龄扑杀时检出法氏囊阳性,雏鹅转染组于转染后2周检出血清阳性。试验进一步对扩增片段进行了BamHⅠ标记位点的检测,并应用GoCV实时荧光定量PCR方法对转染阳性样品进行了定量,结果显示阳性法氏囊组织中病毒含量为1.57×106拷贝/mg,阳性血清含病毒拷贝数在3.52×104～5.92×105拷贝/μL。综上,本试验构建的GoCV全长基因组头尾串联二聚体感染性克隆DNA可以转染鹅胚和雏鹅并增殖出带标记的GoCV克隆。
The full-length genome of goose circovirus (GoCV) was amplified by PCR, and the two genomic sequences were inserted into pGEM-T Easy vector to obtain the full-length genome of GoCV Tail tandem dimer infectious cloning plasmid pGEMT-2GoCV. EcoR Ⅰ digestion linearized with pGEMT-2GoCV, and GoCV negative goose and goose were transfected with liposomes. GoCV was detected by routine PCR and the goose inoculated in the goose in vitro, Positive sera were detected during the week and one of the individuals was positive for bursal disease at 4 weeks of age. In the gosling transfection group, seropositivity was detected 2 weeks after transfection. The BamH Ⅰ marker was further detected in the amplified fragment and the positive samples were quantified by GoCV real-time PCR. The results showed that the virus content in the positive bursal tissue was 1.57 × 106 copies / mg, Positive serum containing virus copies in the 3.52 × 104 ~ 5.92 × 105 copies / μL. In conclusion, the GoCV full-length tandem dimer infectious clone DNA constructed in this study can be used to transfect goose embryos and goslings and propagate labeled GoCV clones.