[Construction of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome].
[Construction of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome].
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DOI：
10.1016/s1872-2075(08)60028-x
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发表时间：
2008-04-01
期刊：
影响因子：
--
通讯作者：
Wang, Mei
中科院分区：
文献类型：
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作者：
Cui, Hongyu;Wang, Yunfeng;Wang, Mei
本研究的目的是构建马立克氏病病毒814株的全基因组，作为感染性细菌人工染色体（BAC）。利用自行设计的选择标记Eco-gpt（1.3kb）和BAC载体pBeloBAC 11（7.5kb）构建了转移质粒pUAB-gpt-BAC 11。将质粒pUAB-gpt-BAC 11与MDV总DNA共转染第二代CEFs，经8轮筛选，获得纯化的重组病毒。提取重组病毒基因组，电穿孔至E. coli中，经酶切和PCR鉴定，获得BAC克隆。最后，我们获得了38个BAC克隆，将来自不同MDV-1 BAC的DNA转染到CEFs中，并通过转染MDV-BAC 2 DNA重建重组病毒。我们成功地克隆了MDV-1814株的全基因组作为感染性细菌人工染色体。利用这些克隆的基因组，成功构建了一个革命性的利用RED/ET重组系统的MDV DNA工程平台，为深入了解MDV基因功能，促进MDV作为外源基因表达载体的应用奠定了基础。此外，它打开了基于BAC产生新型MDV-1疫苗的可能性。
The aim of this study was to construct the complete genome of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.