【摘要】 采用本实验室构建的3种禽传染性支气管炎病毒基因的真核表达质粒pIBVS1p、IBVM、pIBVN,按各50 mg/L配制成质量浓度为150 mg/L的DNA疫苗,经腿部肌肉分点注射免疫1周龄SPF雏鸡。分别在免疫后24 h,73、0及60d,采集试验鸡的血液、心、肝、脾、肺、肾、胸腺、性腺(卵巢/睾丸)及注射部位肌肉进行组织总DNA抽提,以组织总DNA为模板进行PCR扩增。另外,在对照组DNA模板中加入不同拷贝数的质粒,确定PCR反应的灵敏性。以纯化后的组织总DNA为模板,PCR法检测质粒DNA整合到鸡细胞染色体基因组上的可能性,评价疫苗的安全性。结果表明,该疫苗在24 h内迅速分布于全身,并能在血液及所有检测的组织器官内持续分布60 d;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象,证实该DNA疫苗的安全性好。更多还原

【Abstract】 Three IBV plasmid vaccines pIBVS1,pIBVM,pIBVN were constructed in our lab,150 mg/L of IBV DNA vaccine which contains 50 mg/L pIBVS1,50 mg/L pIBVM and 50 mg/L pIBVN were injected intramuscularly into 7-day-old SPF chickens.The blood,heart,liver,spleen,lung,kidney,gonad,thymus and muscle of the injected site were sampled at 24 h,7 d,30 d and 60 d after immunization,for biodistribution and safety study by PCR method.Genome DNA were extracted for PCR assay.The sensitivity of PCR assay was determined by PCR of the control tissue genome after administering different copies of plasmid.Genomic DNA was first purified away from free plasmid using gel electrophoresis and then assayed for integrated plasmid using PCR.The result indicated that IBV DNA vaccine can rapidly distribute in different tissues and blood within 24 h,it can persist in various tissues and blood up to 60 days.The safety experiment indicated that each gel-purified genomic DNA was negative for PCR assay and it was lower than the sensitivity of PCR assay.No genomic integration of DNA vaccine was detected in the immunized chickens.更多还原