摘要
根据猪瘟病毒(HCV)基因组RNA的序列和Ribozyme的结构特点,设计并合成了针对HCV基因组P(80)编码区长为56碱基对(bp)的榔头状HCVRibozyme(HR)基因。将此基因插入质粒pBluescriptSK中,转化XL1-blue细胞,经限制性内切酶鉴定,获得6个阳性克隆pBHR。可用于HCV抗性的体外表达研究。又将羊金属巯因启动子(oMT-promoter)插入pBHR,构建重组质粒poMHR,经序列分析插入的HR基因序列和方向与设计相符。再将oMTHR基因插入质粒pmMT中,最终构成pMHR表达载体,经酶切分析和聚合酶链式反应(PCR)鉴定表为此表达载体中确含oMT启动子和HCV-Ribozyme基因,可用于HCV抗性的体内表达研究。
A hammerhead ribozyme gene of 56 base pairs directed toward P(80) encoding region of genome of hog cholera virus was designed and synthesized based on the genomic RNA sequence of HCV and structural characteristics of ribozymes. With restriction enzyme analysis,six positive clones containing HR gene (pBHR 2,4, 5, 6, 7,8) were obtained by inserting the HCV-Ribozyme gene into plasmid Bluescript SK and transforming ligating products to competent cells of XL-1 blue. The PBHR can be used as a tool for researching the resistant character of the ribozyme against HCV in vitro. Then a recombinant plasmid poMHR was constructed by inserting ovine metallothionein gene promoter into PBHR. The sequence and direction of HCV-Ribozyme gene in poMHR are accordant with the design identified by sequencing. At last, the expression vector PMHR was constructed by inserting oMT-HCV-Ribozyme fusion gene into pmMT. This recombinant plasmid was identified by restriction enzyme analysis and polymerase chain reaction and confirmed containing oMT-promoter and HCV-Ribozyme gene.
出处
《江苏农业学报》
CSCD
1994年第3期34-41,共8页
Jiangsu Journal of Agricultural Sciences
基金
国家863高科技发展计划资助项目