表达猪2型圆环病毒ORF2基因的重组伪狂犬病病毒的构建及鉴定

Construction and Identification of a Recombinant Pseudorabies Virus Expressing ORF2 Gene of Porcine Circovirus Type 2

目的以伪狂犬病病毒为载体,构建表达猪2型圆环病毒ORF2基因的重组病毒,并研究其生物学特性。方法将猪2型圆环病毒ORF2基因插入到伪狂犬病病毒gG缺失通用转移载体中,构建猪2型圆环病毒-伪狂犬病病毒重组中间转移质粒,然后将该质粒与伪狂犬病病毒TK-/gG-/LacZ+基因组共转染IBRS-2细胞,待发生细胞病变后收集病毒液进行重组病毒的筛选及生物学特性测定。结果利用检测PCV2ORF2基因和LacZ基因的PCR方法筛选到重组病毒TK-/gG-/ORF2+,同时用Southernblotting证实外源基因PCV2ORF2已成功插入到TK-/gG-/LacZ+亲本株的基因组中。间接免疫荧光试验(IFA)和Westernblotting结果显示重组病毒中PCV2ORF2基因获得了成功表达。对重组病毒的生物学特性研究表明,重组病毒与其亲本株在不同细胞上的增殖滴度相当,且重组病毒对小鼠无致病性,免疫小鼠后可诱导机体产生抗ORF2蛋白的特异性抗体。结论重组伪狂犬病毒构建成功,且表达的ORF2蛋白具有免疫原性。

[ Objective ] In this paper, a recombinant virus expressing ORF2 gene of PCV2 using pseudorabies virus as vector. [ Method ] The ORF2 gene of porcine circovirus type 2 was inserted into the universal transfer vector deleting gG of pseudorabies virus to generate the recombinant transfer plasmid. The genomic DNA ofPRV TK/gG/LacZ~ strain and pgGORF2 were co-infected into IBRS-2 cells. Then the recombinant virus TK^-/gG^-/ORF2^＋ was selected and its biological characteristic was measured. [Result] The recombinant virus was selected by PCR with ORF2 gene and LacZ gene primers respectively and the PCV2 ORF2 gene had been inserted into the genome of TK^-/gG^-/ORF2^＋ strain identified by Southern blotting. The results of indirect immunofluorescene assay （IFA） and Western blotting indicated that the ORF2 gene was expressed successfully. Propagation of the recombinant virus in cells was corresponding to the parent strain. Animal experiments showed that the recombinant virus TK^-/gG^-/ORF2^＋ was safe to mice and could induce the specific antibodies against expressed ORF2 protein. [ Conclusion ] The recombinant pseudorabies virus was constructed successfully, and the expressed ORF2 protein had immunogenicity.