抗猪瘟病毒Ribozyme基因表达载体的构建

Construction and Sequencing of Vectors Expressing a Ribozyme Gene Against Hog Cholera Virus

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.