禽腺联病毒的分离及其基因组鉴定  

Isolation and genome identification of avian adeno-associated virus

作　　者：王建业[1] 孙怀昌[1] 朱国强[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2005年第2期7-10,共4页Journal of Yangzhou University：Agricultural and Life Science Edition

摘　　要：为了获得用于重组载体构建的禽腺联病毒(AAAV),用无菌处理后的鸡粪便样品与鸡胚致死孤儿病毒同时接种SPF鸡胚,根据已发表的AAAV基因序列设计引物,用PCR对致死鸡胚的尿囊液进行检测,结果从1份样品接种的鸡胚尿囊液中扩增到预期大小的AAAVRep和Cap基因片段;对纯化后的PCR产物进行序列测定,将所获序列与已发表的AAAVRep和Cap基因进行比较,其核苷酸序列同源性分别为97.1%和94.6%;将PCR检测阳性的尿囊液进行鸡胚传代,用PCR对不同时间死亡鸡胚的尿囊液进行再次检测,结果均扩增到预期大小的基因片段;提取病毒核酸,电泳观察到约4.7kb的AAAV基因组。说明已成功地从鸡粪便样品中分离到1株AAAV,为重组AAAV载体的构建打下了基础。<Abstrcat> To obtain avian adeno-associated virus (AAAV) for construction of the recombinant vectors, ten aseptically treated fecal samples from 7-week-old chickens were co-inoculated with the type 1 fowl adenovirus (CELO) into 11-day-old embryonated eggs of SPF chickens. Their allantoic fluids were harvested at different time points of pos- tinoculation and submitted to PCR amplification using primes designed according to the Rep and Cap genes of the standard AAAV VR-865 strain. The results showed that two expected gene fragments of 485 bp and 453 bp were detected from the allantoic fluid of the chicken embryo inoculated with one fecal sample. The sequence analysis showed that the two gene fragments were 97.1% and 94.6% identical to the Rep and Cap genes of the AAAV VR-865 strain. The isolated virus was successfully passed in embryonated SPF eggs based on PCR detection of their egg allantoic fluids. Following extraction of the nucleic acid from ultracentrifugated virions, an expected size of 4.7 kb of AAAV genome was detected by agarose gel electrophoresis. These results indicate that an AAAV isolate has been successfully isolated from a normal chicken fecal sample.

关 键 词：基因组 分离 鉴定 禽 基因片段 SPF鸡胚 鸡胚尿囊液 PCR产物 序列同源性 PCR检测 载体构建 无菌处理 序列设计 序列测定 病毒核酸 Cap Rep 样品 核苷酸 重组 接种 粪便 预期 行比 

分 类 号：S858.3[农业科学—临床兽医学] Q78[农业科学—兽医学]