表达O型口蹄疫病毒 VP1基因的重组病毒BHV-1的构建与鉴定

Construction and identification of recombinant BHV-1 expressing foot and mouth disease virus VP1 gene

摘要:

摘要：【目的】为了构建表达口蹄疫病毒（O/China/99）VP1基因的牛疱疹病毒1型，将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒（CMV）启动子之下构建gE基因缺失转移载体。【方法】利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过筛选白色病毒蚀斑，得到重组病毒BHV-1/gE-/VP1。【结果】PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中，间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。【结论】本研究成功的构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1，为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。

关键词:关键词： 口蹄疫病毒;VP1基因;牛疱疹病毒1型;磷酸钙介导转染

Abstract:

Abstract: [Objective]: In order to construct the recombinant bovine hepervirus-1(BHV-1) which expressed foot and mouth disease virus (FMDV)VP1 gene, we constructed a BHV-1 gE gene transfer vector by inserting the synthetic VP1 gene of FMDV (O/China/99) under the immediate-early promoter of cytomegalovirus. [Methods]: The mixtures of parental virus (BHV-1/gE-/LacZ+ ) DNA and transfer vector was transfected into bovine turbinate cells using calcium phosphate-mediated transfection. Then the propagated viruses were harvested. The recombinant BHV-1 (designated BHV-1/gE-/VP1) was obtained by selection for white virus plaques. [Results]: PCR results showed that VP1 gene was successfully inserted into the genome of BHV-1/gE-. The expression of VP1 in infected cells was proved by indirect immunofluorescence assay and Western blotting. [Conclusion]: The research provided a basis for development of BHV-1 vector vaccines for FMD and other important bovine infectious diseases.

Key words:Keywords: FMDV; VP1 gene; Bovine hepervirus-1; Calcium phosphate-mediated transfection