猪瘟病毒反义cDNA片段的化学合成及克隆

CHEMICAL SYNTHESIS AND CLONING OF HOG CHOLERA VIRUS ANTISENSE cDNA FRAGMENTS

In order to evaluate the inhibition effect of specific antisense RNA expressed in vivo on the replication of hog cholera virus in cells, 3 fragments of the virus cDNA were chemically synthesizd according to the sequence of the genome of Alfort strain virus and subsequently cloned into BamH I site of plasmid pUC19 and M13 cloning vector. The correctness of these fragments was confirmed by DNA sequence analysis. Fragment A ( 102bp ) and fragment B ( 45bp ) span the region of promoter and the part of downstream coding sequence including ATG.Fragment C(99bp ) is a part of sequence coding for the important helicase and protease. All these 3 fragments were selected in consideration of the fact that anti sense inhibition occurs most effectively either in the initiation region of protein translation on mRNA strand including AUG and its upstream ribo some binding site or in the regions coding for important proteins. Two antisense RNA expressing vectors specific for the virus have been constructed by insertion of fragment A and B respectively in reverse orientation between mouse metallothionein-I promoter and polyadenylation signal sequence.