| 摘要: |
| 【目的】利用腺病毒pAdEasy-1表达系统构建含山羊Δfosb基因的重组腺病毒，为在原代细胞中研究该基因的功能奠定基础。【方法】扩增山羊的Δfosb基因，与pAdTrack连接构建pAdTrack-HA-Δfosb。用pAdTrack-HA-Δfosb转化含有pAdEasy-1的BJ5183细胞，同源重组后获得重组腺病毒质粒pAd-HA-Δfosb，用其转染HEK293细胞，包装得到重组腺病毒。对重组腺病毒进行PCR和Western blot鉴定。【结果】酶切鉴定、绿色荧光蛋白观察及PCR均证明，重组腺病毒质粒构建正确；Western blot检测到了Δfosb蛋白的表达。【结论】成功构建获得了可表达Δfosb蛋白的重组腺病毒rAd-HA-Δfosb。 |
| 关键词: 山羊 Δfosb 重组腺病毒 |
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| 基金项目:西北农林科技大学基本科研业务费（Z109021002）；农业部公益性行业科研专项经费项目(3-45) |
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| Construction of recombinant adenoviral vector of Δfosb gene in goat |
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| Abstract: |
| 【Objective】The study was to construct recombinant adenovirus vector bearing the goat Δfosb gene and to amplify the adenovirus in HEK293 cells，which would provide a basis for further study on the role of Δfosb gene in primary culture cells.【Method】Inserting the gene fragments containing both the complete CDS region of goat Δfosb gene and HA tag into adenovirus shuttle vector pAdTrack,a recombinant shuttle vector pAdTrack-HA-Δfosb was constructed.After identifying by digestion and sequencing,pAdTrack-HA-Δfosb was linearized with PmeⅠ,and then it was homologously recombined with plasmid pAdEasy-1 containing the adenovirus backbone DNA in BJ5183 E.coli cells to reform a new recombinant pAdEasy-HA-Δfosb.The DNA extracted from digested pAdEasy-HA-Δfosb by PacⅠ was transfected into HEK293 cells by liposomes method.Then the integrite adenovirus pAd-HA-Δfosb was packaged in HEK293 cells.【Result】It was confirmed by PCR detection and Western blot that the recombinant adenovirus rAd-HA-Δfosb contained Δfosb gene and could express both goat Δfosb gene and HA tag in HEK293 cells.【Conclusion】The recombinant adenovirus rAd-HA-Δfosb was constructed successfully. |
| Key words: goat Δfosb recombinant adenovirus |
