EIAV标记感染性克隆分子的构建

Construction of an EIAV Infectious Molecular Clone with Six HISTag

应用已构建好的EIAV驴白细胞弱毒疫苗株的感染性分子克隆载体(pOK8266)为模板,通过SOE PCR方法在感染性分子克隆载体的S2基因独特区内引入突变点,形成含有酶切位点(NspV)的突变体(P1P4)。将突变体(P1P4)亚克隆至pB luescript SK(M13-)质粒,形成pB-P1P4中间载体;人工合成两条6个组氨酸的引物,作为标签,将其插入pB-P1P4中间载体的NspV位点,形成带标签的中间载体;用ApaI酶切pOK8266和中间载体,连接转化,构建带标签的重组感染性分子病毒克隆载体(EIAV-p8.2-H IS),经PCR、酶切和测序表明,6个组氨酸已插入pOK8266的S2基因内。分别转染驴皮肤细胞(FDD)和驴白细胞,将转染驴皮肤细胞收获物传至第六代,电镜下未观察到EIAV病毒;而将转染驴白细胞收获物传代至第6代时,电镜下观察到了典型的病毒粒子。提取前病毒DNA,PCR扩增P1P4片段,亚克隆至pMD18-T载体,测序证明前病毒DNA含有6个组氨酸,从而在体外获得了感染性克隆病毒株。本研究通过SOE法巧妙地对S2基因引入突变,插入H IS标签,成功地获得了标记感染性分子克隆,证明S2基因缺失突变株在体外巨嗜细胞上培养不影响其复制力,为野毒株和疫苗毒的鉴别诊断打下基础。

Equine infectious anemia virus （ EIAV ） is a member of the lentivirus of retrovirusae and the causative agent of equine infectious anemia （ EIA）. The Chinese donkey leukocyte attenuated strain of EIAV （ DLA） was the only successful lentivirus vaccine up to now, which has been applied extensively in China against EIA. EIAVDLA can be served as a model for study on lentivirus. Because of difficulty in differentiating EIAV wild strain from the vaccine strain （DLA） in vivo, we have to build a fast identification method between wild strain and vaccine strain of EIAV. S2 is an accessory protein of equine infectious anemia virus （ EIAV ） , the S2 function is not fully defined. It has been reported that the EIAV S2 is not essential and does not appear to affect virus infection and replication in vitro. Thus, we introduced a HIS-tag into the S2 gene of an EIAV infectious molecular clone recombinant plasmid （ pOK8266 ） by using SOE PCR method and obtained a new recombinant plasmid with HIS - tag, designated as EIAV - p8.2 - HIS. The complete nucleotide sequence of EIAV - P8.2 - HIS was determined. The EIAV -P8.2 -HIS was transfected into the donkey leukocyte culture. Following an incubation period, reverse transcriptase activity was detected in cell culture supernatants. Cytopathic effect was observed by No. six passages post-infection in donkey leukocyte infected by the virus derived from EIAV molecular clones EIAV-POK8.2-HIS. EIAV virions were observed by electron microscope from donkey leukocytes infected by virus derived from EIAVPOK8.2-HIS. These results demonstrate that changing the EIAV S2 gene （ inserting small foreign gene fragment）does not appear to affect replication properties in target cells in vitro. Furthermore, infectious molecular clone of DLA-EIAV strain with His-tag lay the foundation for differentiating EIAV wild strain from donkey leukocyte attenuated strain of EIAV （ DLA ） vaccine strain.