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Xba I／BamH I和BamH I／Kpn I双酶切之后，与Xba I／Kpn I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-Ceu I和PbSce I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BDAdeno-X Virus 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24-48h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．
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 Xba I /Barn I and BamH I/ Kpn I ,respectively. These gene fragments were ligated into pshuttle2 vector digested with Xba I/Kpn I , 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-Ceu I and PI-Sce I and ligated with BD Adeno-X Virus DNA in vitro. Ligating products were then thansformed into DH5a competent cell. Two re- combinant adenovirus plasmids were confirmed by PCR, Pac I digestion and sequencing, respectively,which were named as A-P12a3c and A-P12a3abc. After digestion with Pac I 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.
出处
《甘肃农业大学学报》
CAS
CSCD
2008年第4期17-22,共6页
Journal of Gansu Agricultural University
基金
国家重大基础研究发展规划“973”资金资助(2005CB523201)
