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A型口蹄疫病毒多基因重组腺病毒载体的构建
引用本文:孙普,卢曾军,刘霞,李平花,白兴文,郭建宏,包慧芳,刘在新.A型口蹄疫病毒多基因重组腺病毒载体的构建[J].甘肃农业大学学报,2008,43(4).
作者姓名:孙普  卢曾军  刘霞  李平花  白兴文  郭建宏  包慧芳  刘在新
作者单位:1. 甘肃农业大学动物医学院,甘肃,兰州,730070;中国农业科学院兰州兽医研究所,国家家畜疫病病原生物学重点实验室,农业部畜禽病毒学重点实验室,国家口蹄疫参考实验室,甘肃,兰州,730046
2. 中国农业科学院兰州兽医研究所,国家家畜疫病病原生物学重点实验室,农业部畜禽病毒学重点实验室,国家口蹄疫参考实验室,甘肃,兰州,730046
基金项目:国家重点基础研究发展计划(973计划)
摘    要:采用体外连接法构建了含有A型口蹄疫病毒(FMDV)P1-2A和3C或3ABC基因的重组腺病毒表达载体.利用PCR技术分别扩增得到P1-2A、3C和3ABC基因,经XbaⅠ/BamHⅠ和BamHⅠ/KpnⅠ双酶切之后,与XbaⅠ/KpnⅠ双酶切的穿梭载体pShuttle2大片段连接,获得重组穿梭质粒pSh-P12a3c和pSh-P12a3abc.然后用I-CeuⅠ和PI-SceⅠ双酶切穿梭质粒回收目的基因表达盒,通过体外连接法将目的基因表达盒与BD Adeno-X Vi-rus DNA连接,转化DH5α感受态菌.获得两个重组腺病毒质粒分别命名为A-P12a3c和A-P12a3abc,经PCR、酶切及测序鉴定正确.用PacⅠ酶切后转染HEK293细胞,取转染细胞裂解液上清连续传至第5代时,在24~48 h内细胞完全病变.分别提取第3、7代病毒DNA,可扩增出目的基因,表明目的基因已整合到腺病毒基因组内,且能稳定传代.

关 键 词:口蹄疫病毒  腺病毒载体  体外连接

Construction of recombinant adenovirus vector containing the capsid and 3C protease or 3ABC coding regions of foot-and-mouth disease virus
SUN Pu,LU Zeng-jun,LIU Xia,LI Ping-hua,BAI Xing-wen,GUO Jian-hong,BAO Hui-fang,LIU Zai-xin.Construction of recombinant adenovirus vector containing the capsid and 3C protease or 3ABC coding regions of foot-and-mouth disease virus[J].Journal of Gansu Agricultural University,2008,43(4).
Authors:SUN Pu  LU Zeng-jun  LIU Xia  LI Ping-hua  BAI Xing-wen  GUO Jian-hong  BAO Hui-fang  LIU Zai-xin
Abstract:Two recombinant replication-defective human adenovirus serotype 5 expression vectors containing FMDV serotype A capsid P1-2A and viral 3C protease or 3ABC coding region were constructed by in vitro ligation.PCR product of P1-2A,3C and 3ABC were digested with XbaⅠ/BamⅠ and BamHⅠ/KpnⅠ,respectively.These gene fragments were ligated into pshuttle2 vector digested with XbaⅠ/KpnⅠ,and obtained recombinant plasmid pSh-P12a3c and pSh-P12a3abc.The target gene cassettes were excised from above two recombinant shuttle vectors by digesting with I-CeuⅠ and PI-SceⅠ and ligated with BD Adeno-X Virus DNA in vitro.Ligating products were then thansformed into DH5α competent cell.Two recombinant adenovirus plasmids were confirmed by PCR,Pac I digestion and sequencing,respectively,which were named as A-P12a3c and A-P12a3abc.After digestion with PacⅠ the linerized recombinant adenovirus plasmids were transfected into HEK293 cells for package of recombinant adenovirus.The cell pathogenicity effect(CPE) could be observed within one week after transfection,and complete CPE appeared within 24~48 h after five round passages.PCR demonstrated that the target genes were inserted into adenovirus DNA.
Keywords:FMDV  adenovirus vector  in vitro ligation
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