Untranslated region-specific artificial microRNAs that effectively inhibit the replication of different strains of porcine reproductive and respiratory syndrome virus
A technology for respiratory syndrome and virus replication, applied in the fields of genetic engineering and biological product research, can solve the problems of attenuated vaccine loose virus and inaccurate immune effect of inactivated vaccine, etc., and achieve the effect of inhibiting the replication of heterologous strains
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[0035] 1. Construction of artificial miRNA expression vector
[0036] Reference to the 5'miR flanking sequence (134-160 nucleotides) and 3'miR flanking sequence (221-265 nucleotides) in the mouse BIC non-coding mRNA sequence (GenBank ACCESSION AY096003) and the herpes simplex virus TK gene (GenBank ACCESSION HQ686001) polyA sequence (1115-1136 nucleotides), designed an artificial miRNA expression cassette, introduced HindIII and DraIII sites at both ends, and introduced two BbsI sites (5-GTGTCTTCAAGATCTGGAAGACAT -3, SEQ ID NO: 5). The sequence was sent to Nanjing GenScript Biotechnology Co., Ltd. for chemical synthesis, and the synthetic sequence was cloned into the pMD-18 vector. The miRNA expression cassette was excised from the synthetic vector with restriction enzymes HindIII and DraIII, and connected with the eukaryotic expression vector pcDNA3 (Invitrogen Company) that removed the multiple cloning site and BGH polyA sequence by isoenzyme digestion, and obtained the expr...
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