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Process for producing small RNA recombinant birds adeno-associated virus for expression of infectivity resistant bursa of fabricius disease virus

A technology of RNA recombination and bursal disease, applied in the field of genetic engineering and biological product research

Inactive Publication Date: 2009-04-08
YANGZHOU UNIV
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AI Technical Summary

Problems solved by technology

Avian adeno-associated virus (AAAV) has similar latent infection characteristics and genome structure to mammalian AAV, and it has been proven that it can stably express foreign genes in cells of avian origin, but there is no report of using AAAV to deliver anti-avian virus siRNA or miRNA

Method used

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  • Process for producing small RNA recombinant birds adeno-associated virus for expression of infectivity resistant bursa of fabricius disease virus
  • Process for producing small RNA recombinant birds adeno-associated virus for expression of infectivity resistant bursa of fabricius disease virus
  • Process for producing small RNA recombinant birds adeno-associated virus for expression of infectivity resistant bursa of fabricius disease virus

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Embodiment Construction

[0027] Vector source:

[0028] pRFPRNAiC vector: introduced from ARK-GENOMICS, UK. (Literature: Das, R. Metal. Developmental Biology, 2006, 294: 554-563)

[0029] pCR-AAAV vector: constructed and preserved in Sun Huaichang Laboratory of Yangzhou University. (Document: Wang Jianye et al. Journal of Yangzhou University (Agriculture and Life Science Edition), 2005, 26: 1-4)

[0030] pcDNA-ARC: Constructed and preserved by Sun Huaichang Laboratory of Yangzhou University. (Wang, A.P. et al. Chinese Journal of Virology, 2007, 23:292-297)

[0031] pEGFP-N1: introduced from BD Biosciences Clontech, USA. (GenBank Accession #U55762; Catalog #6085-1; PT3027-5)

[0032] pHelper: introduced from Stratagene Company of the United States. (Document: Matsushita, T., et al. GeneTherapy, 1998, 5: 938-945)

[0033] The specific operation steps are as follows:

[0034] 1. Selection of siRNA:

[0035]Use the siRNA TargetFinder software and methods of GenScript Corporation (http: / / www.gensc...

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Abstract

The invention relates to a preparation method of establishing a reconstructed aviadenovirus for expressing infectious bursal disease virus (IBDV) miRNA, pertaining to the research field of gene engineering and biological product, comprising the selection of siRNA, the synthesis and cloning of double-stranded short-barrette oligonucleotide, the construction of virus transfer vector containing miRNA expression cassette aviadenovirus, the generation and identification of reconstructed virus expressed, the detection of the duplication function by miRNA to IBDV expressed by the reconstructed virus and the inhibition function to genetic expression. Compared with the prior art, the method can not only effectively and persistently express miRNA in bird cells, has specific and persistent inhibition to the duplication of virus and expression of gene of IBDV, but also has not infective function to poultry, which can be applied to in vitro experiments as well as be developed as a novel anti-avain-virus biological product.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering and biological product research. In particular, it relates to a preparation method for expressing anti-infectious bursal disease virus microRNA recombined avian adeno-associated virus. Background technique: [0002] RNA interference (RNA interference, RNAi) is a sequence-specific post-transcriptional gene silencing technique that has developed rapidly in recent years, and has a wide range of applications in functional genomics research, new drug development, cancer treatment, and antiviral infection. In terms of antiviral infection, existing research data show that RNAi can almost inhibit the replication of all viruses (including DNA and RNA viruses) to varying degrees. [0003] Interfering RNAs with RNAi effects include short interfering RNAs (siRNAs) and microRNAs (miRNAs). Currently, the methods for obtaining siRNA or miRNA mainly include chemical synthesis, in vitro transcription and vect...

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Application Information

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IPC IPC(8): C12N7/01C12N15/11C12N15/86
Inventor 孙怀昌王永娟沈鹏鹏张鑫宇夏晓莉
Owner YANGZHOU UNIV
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