猪圆环病毒2型核酸疫苗的小鼠免疫保护试验  

Experiment on immune protection of DNA vaccine against porcine circovirus 2 in mice

作　　者：张宁[1] 侯韶毅[1] 韦英益[1] 李茂宁[1] 肖爱欢[1] 谢丽华[2] 刘芳[1] 高永锐 卢桂娟 陆有飞 黄伟坚[1] 

机构地区：[1]广西大学动物科技学院,广西南宁530005 [2]广西梧州市动物疫病预防控制中心,广西梧州543002 [3]广西丽原生物股份有限公司,广西南宁530001

出　　处：《畜牧与兽医》2010年第1期11-16,共6页Animal Husbandry & Veterinary Medicine

基　　金：广西科学研究与技术开发项目(桂科攻0537008-3);广西科学研究与技术开发项目(桂科攻0537022-1);广西科技基础条件平台建设示范研究项目(桂科能0630006-6B)

摘　　要：为了研究猪圆环病毒2型(PCV2)核酸疫苗在小鼠攻毒试验中的免疫保护效果,以PCV-2 GXWZ-1株为模板,扩增出ORF2基因及其截短基因8个片段(A(ORF2)、B(51-100aa)、C(101-150aa)、D(181-235aa)、E(151-200aa)、F(51-150aa)、G(101-235aa)、H(51-235aa)),将其插入到pcDNA3.0载体中,构建出真核表达质粒,并将其转染至PK-15细胞,用间接免疫荧光试验检测其瞬时表达情况。将试验小鼠随机分成9组,其中免疫组7组,阴阳性对照各1组,将纯化的真核表达质粒对小鼠进行组合免疫;二免后,用经处理过的PCV-2 GXWZ-2株阳性病料悬液腹腔注射免疫组和非免疫对照组小鼠,阴性对照组用生理盐水腹腔注射;其后进行体重记录、病理切片制作及PCR检测。结果表明:共有6个真核表达质粒成功在PK-15细胞中表达。在攻毒后的3周内,阳性对照组小鼠PCR诊断均为阳性;免疫组中,部分组小鼠在攻毒后第1周或在第2周为阳性,到第3周时各免疫组小鼠全部为阴性;阴性对照组始终为阴性。免疫组在病理保护学方面明显优于非免疫对照组,非免疫对照组的体重增长速率略低于免疫组和阴性对照组。由此可见猪圆环病毒2型核酸疫苗在小鼠攻毒试验中有明显的保护作用。To study immune efficacy of DNA vaccines with truncated ORF2 gene of porcine circovirus 2 （ PCV-2）, the ORF2 associated gene fragments, including A （complete ORF2）, B （51-100aa）, C （101-150aa）, D （181-235aa）, E （151-200aa）, F （51-150aa）, G （101-235aa） and H （51-235aa） , were amplified from PCV-2 GXWZ-1 strain by PCR and cloned into pcDNA3.0 vector to construct the eu- karyotic expression plasmids pcDNA-A, pcDNA-B, pcDNA-C, peDNA-D, pcDNA-E, pcDNA-F, pcDNA-G and pcDNA-H. The eight purified recombinant plasmids were transfeeted into PK-15 cells and their expressed products were examined by indirect immunofluorescenee assay. Experimental mice were randomly divided into 9 groups, including 7 immunity groups, positive and negative control groups. The mice in the immunity groups were m. i. vaccinated with the purified expressed plasmid. After twice vaccination, the immunity groups and positive control were p.i. injected with the suspension of PCV-2 GXWZ-2 strain, and negative control with physiological saline. Weight record and pathological observation and PCR amplification were performed. The results demonstrated that fluorescence could be observed in the cells transfected with pcDNA-B, pcDNA-C, pcDNA-D, pcDNA-E, pcDNA-F and pcDNA-G. In the three weeks after challenge, some mice in the immunity groups were positive in the first or second week, and all were negative in the third week. Pathological observation revealed that the lesions in the immunity groups were less than those in the positive control. Body weight growth rate in the positive control was lower than the two other groups. It suggested that the DNA vaccines provided the distinct protection against PCV-2 in the vaccinated mice.

关 键 词：ORF2截短基因 重组质粒 间接免疫荧光试验 组合免疫 免疫保护效果 

分 类 号：S852.4[农业科学—基础兽医学]