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

Inactive Publication Date: 2011-12-28
YANGZHOU UNIV
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Problems solved by technology

At present, the disease is mainly prevented by vaccination, but the immune effect of the existing inactivated vaccine is not reliable, and the attenuated vaccine has the risk of spreading the virus, so it is urgent to study new strategies for the prevention and control of the disease

Method used

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  • Untranslated region-specific artificial microRNAs that effectively inhibit the replication of different strains of porcine reproductive and respiratory syndrome virus
  • Untranslated region-specific artificial microRNAs that effectively inhibit the replication of different strains of porcine reproductive and respiratory syndrome virus
  • Untranslated region-specific artificial microRNAs that effectively inhibit the replication of different strains of porcine reproductive and respiratory syndrome virus

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Experimental program
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Embodiment

[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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Abstract

The invention designs an untranslated region specific artificial micro RNA (miRNA) capable of effectively inhibiting replication of porcine reproductive and respiratory syndrome (PRRS) virus strains, which belongs to the field of researches on gene engineering and biological products. The sequence of the miRNA is represented by one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. The invention also discloses the construction of an artificial miRNA expression vector based on RNA polymerase II, design of miRRNAi, synthesis and cloning of double-stranded oligo, detection of expression of miRNA and inhibition effect on replication of different PRRSV strains. Compared with siRNAs for genes of other PRRSVs, the artificial miRNA screened by the invention, according to the conservative sequence of the untranslated region, can inhibit the replication of homologous virus strains and heterologous virus strains and can be used in both the development of anti-PRRSV infection preparations and breeding transgenic pig disease resistance lines.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biological product research. It specifically relates to the preparation and use of non-translated region-specific artificial microRNAs that effectively inhibit the replication of different strains of porcine reproductive and respiratory syndrome virus (PRRSV). Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is an important infectious disease that endangers the swine industry in the world. It is mainly characterized by reproductive disorders such as fever, abortion, stillbirth, and mummified fetuses in sows, respiratory symptoms and high mortality in piglets. At present, the disease is mainly prevented by vaccination, but the immune effect of the existing inactivated vaccines is not reliable, and the attenuated vaccine has the risk of spreading the virus, so it is urgent to study new strategies for the prevention and control of the disease. [0003] RNA int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
Inventor 孙怀昌夏冰宋红芹陈阳张鑫宇夏晓莉
Owner YANGZHOU UNIV
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