摘要
本研究将以vvIBDV GX8/99株4株克隆化细胞毒为研究对象,通过鸡胚成纤维细胞连传至20代,得到4株克隆化毒F20代:GX-IBDVE10C25CL1-20、GX-IBDVE10C25CL2-20、GX-IBDVE10C25CL4-20、GX-IBDVE10C25CL5-20。分别提取RNA,通过Nested-PCR、基因克隆、核苷酸序列测定和分析,从而摸索出了vvIBDV GX8/99株四株克隆株在细胞传代过程中VP2核苷酸序列和推导出的氨基酸序列的变化规律。与原始囊毒和D78疫苗毒相比,4个细胞适应毒克隆株F20 VP2高变区的约145个氨基酸中,有12个位点发生了相同的变异,变得与适应细胞的疫苗毒D78株基本一致。实验表明VP2高变区大多数氨基酸的变异与对细胞培养或组织的亲和性的关系更为密切。将4株克隆毒株的20代细胞毒免疫SPF鸡,40日龄进行攻毒实验,免疫效果明显,保护率几乎达到100%,二免后第14 d左右抗体浓度达到高峰。从抗体水平和病理组织学变化看,4株克隆化毒F20代中GX-IBDVE10C25CL4-20株免疫效果稳定,病理变化不明显,有望成为理想的疫苗株,为控制vvIBDV的感染提供一条新的途径。
The study is based on 4 attenuated cloning virus strains of vvIBDV GX8/99,which were trained in CEF to 20 generations.Try to find out the evolution of four strains of cloning＇s nucleotide sequences and amino acids during the process of attenuation,by Polymerase chain reaction（PCR）,gene cloning of four strains of cloning,Compared with the initial bursa virus,12sites in 145 amino acids of high variable region of VP2 from four viruses changed the same.The chickens were immunized by four strains viruses in the age of 14d,immune effect is obvious,had good immune protects.On 14 days after the second inoculation,antibody concentration is highest.Frem the pathological and histological change,experiments show that the variation of VP2 region associate with immunogenicity,and had something to do with cell culture or tissue appetency.Four strains viruses had transitory tissue damnification,It will get better immune effect in appropriate immune time.The strain GX-IBDVE10C25CL4-20 is expected to become the ideal vaccine strains.It may offer a new way in controlling the infection of vvIBDV.
作者
王新建
朱瑞良
谭燕玲
魏凯
王慧
王尊民
赵庆友
赵昌亮
WANG Xin-jian;ZHU Rui-liang;TAN Yan-ling;WEI Kai;WANG Hui;WANG Zun-min;ZHAO Qing-you;ZHAO Chang-liang(College of Animal Science and Veterinary Medicine,Shandong Agricultural University,Taian 271018,China)
出处
《山东农业大学学报（自然科学版）》
CSCD
北大核心
2012年第1期55-61,共7页
Journal of Shandong Agricultural University：Natural Science Edition
基金
山东省科技攻关项目(2008GG10009023)
农业部公益性行业科研专项(#200803019)
