共表达猪瘟病毒E0、E2基因重组鸡痘病毒的遗传稳定性

Genetic stability of recombinant fowlpox virus co-expressing E0、E2 gene of classical swine fever virus

【目的】研究共表达猪瘟病毒E0、E2基因的重组鸡痘病毒rFPV-E0-E2的遗传稳定性。【方法】将重组鸡痘病毒rFPV-E0-E2在鸡胚成纤维细胞上连续传20代,取第5、10、15和20代重组病毒进行以下检测:用蓝斑试验鉴定各代重组病毒纯度;用PCR方法扩增猪瘟病毒E0、E2基因,进行基因序列分析;用间接免疫荧光实验检测各代重组病毒中猪瘟病毒E0、E2基因的表达。【结果】各代次重组病毒产生的蚀斑均为蓝色,表明病毒纯度稳定。从各代重组病毒DNA中均扩增出了约700 bp和1 200 bp的条带,分别与E0和E2基因片段长度一致,基因序列分析发现,第5、10、15代重组鸡痘病毒中的E0、E2基因序列与原始转移载体序列完全一致,第20代重组病毒插入基因有2处发生了点突变(即E0基因139位A→G,E2基因413位A→G),其编码的氨基酸也发生了相应变化(即E0蛋白47位Thr→Ala,E2蛋白138位Glu→Gly),但蛋白抗原表位未发生明显的变化。间接免疫荧光实验检测到各代重组病毒均能正常表达目的蛋白。【结论】重组鸡痘病毒rFPV-E0-E2在鸡胚成纤维细胞上传20代以内,具有良好的遗传稳定性,插入的猪瘟病毒E0、E2基因能够正确稳定表达。

[Objective] The aim of this study is to evaluate the genetic stability of the recombinant fowl poxvirus （rFPV） expressing E0 and E2 gene of Shimen strain of classical swine fever virus （CSFV）. [Method] The recombinant fowl poxvirus rFPV-E0-E2 was serially passaged for 20 passages in specificpathogen-free chicken embryo fibroblast （CEF） culture. The passage 5,10,15 and 20 were chosen to identify by blue plaque assay,PCR amplification,gene sequence analysis and indirect immunofluoresence assay. [Result] The blue plaque assay showed that these rFPVs were pure with plaque blue. From all of these rF- PV-E0-E2, E0 and E2 gene can be amplified, and there were not any changes on the nucleotide and in passages 5,10 and 15,only one point mutation on E0 gene（139A→G）and another on E2 gene（413A→G）in passages 20. Point mutations caused two amino acid changes, E0 protein（47 Thr→Ala）and E2 protein （138 Glu→Gly）, but no obvious changes on epitopes. Then expressions of E0 and E2 genes the rFPV were identified by indirect immunofluoresence assay. [Conclusion] All the results showed that the rFPV has good genetic stability within 20 passages.