摘要
马传染性贫血(EIA)弱毒疫苗的广泛使用存在野毒和疫苗毒鉴别困难的问题。本研究以已构建的马传染性贫血驴白细胞弱毒疫苗株的感染性分子克隆(pOK8266)为基础,在其S2基因内引入NspV酶切位点,将人工合成的编码6个组氨酸的寡核苷酸插入NspV位点,获得带有组氨酸标签的重组质粒pOK8266_HIS。将pOK8266_HIS转染驴白细胞,将驴白细胞转染产物传至第6代时,在电镜下观察到了典型的马传染性贫血病毒粒子。提取pOK8266_HIS衍生病毒的前病毒基因组DNA,通过PCR扩增和测序表明,衍生病毒基因组中引入了6个组氨酸标签,从而获得了带有分子标志的马传染性贫血弱毒疫苗株,为野毒株和疫苗病毒的鉴别诊断奠定了基础。本研究还证明了S2基因中的插入突变并不影响马传染性贫血病毒的体外复制。
Equine infectious anemia virus (EIAV) donkey leukocyte attenuated vaccine has been applied extensively in China,but a method being able to differentiate wild_type EIAV strains from the vaccine strain is not available.In this study,an NspV site was introduced into the S2 gene of pOK8266, an infectious molecular clone derived from EIAV vaccine virus,and then a 6_histidine tag was introduced into NspV site,generating pOK8266_HIS.The pOK8266_HIS was transfected into donkey leukocytes.Cytopathogenic effects were found in donkey leukocytes infected with the virus rescued from pOK8266_HIS after six passages.EIAV virions were observed by electron microscope.Sequence analysis indicated that the proviral DNA of the rescued virus contains the histidine tag.The results also demonstrated that insertion of short sequence in S2 gene does not affect in vitro replication of EIAV in target cells.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2005年第3期193-195,共3页
Chinese Journal of Preventive Veterinary Medicine
基金
"863计划"(2001AA223041)
国家自然科学基金(30170706)资助.
