Method for screening non-essential regions for replication of goat pox virus and universal transfer vectors for same

A technology for replicating non-essential regions and goat pox virus, applied to viruses/bacteriophages, using vectors to introduce foreign genetic material, biochemical equipment and methods, etc., can solve problems such as reducing virus pathogenicity, and achieve better safety Effect

Inactive Publication Date: 2011-09-07
广西壮族自治区动物疫病预防控制中心
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Problems solved by technology

Therefore, we speculate that the two genomic regions of GTPV ORF5-ORF19 and ORF149-ORF151 may be non-essential regions for virus replication, whic

Method used

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  • Method for screening non-essential regions for replication of goat pox virus and universal transfer vectors for same
  • Method for screening non-essential regions for replication of goat pox virus and universal transfer vectors for same
  • Method for screening non-essential regions for replication of goat pox virus and universal transfer vectors for same

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Embodiment Construction

[0044] 1 Primer design

[0045] (1) Copy non-essential area on the left (ORF5~ORF19)

[0046] The left arm of homologous recombination (LL): the upstream primer is 5'-AATTTTACAGCTGAGCAA TC-3', and the downstream primer is 5'-ACGAAGGATTTAATTCAACA-3'. The amplified fragment with a span of 1.66 kb was located in the ORF3 to ORF6 region on the left side of the GTPV genome (refer to the full sequence of the Pellor strain GTPV genome of Genbank accession number, located at 1281nt~2941nt). The right arm of homologous recombination (LR): the upstream primer is 5'-TACTGGGTTGTTGAAT GGTT-3', and the downstream primer is 5'-AAATCTGAATGGGCTAGAAA-3'. Amplified a fragment with a span of 1.55kb, located in the region from ORF19 to ORF20 on the left side of the GTPV genome (located at 12316nt~13865nt)

[0047] (2) Copy non-essential area on the right side (ORF149~ORF151)

[0048] The left arm of homologous recombination (RL): the upstream primer is 5'-TTTCTTATCCGCATTAGA TG-3', and the downstream pri...

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Abstract

The invention relates to a method for screening non-essential regions for replication of a goat pox virus and universal transfer vectors for same. The method comprises the steps of amplifying two-end gene segments of any two regions of a goat pox virus gene by using a PCR (Polymerase Chain Reaction) method; then, inserting an enhanced green fluorescent protein (EGFP) gene and a xanthine-guanine phosphoribosyl transferase (gpt) gene expression cassette into the segments; establishing two universal transfer vectors of the goat pox virus; and acquiring a recombinant virus expressing an exogenous gene stably from the transfer vectors, thereby determining the selected regions to be non-essential regions for replication of the goat pox virus, wherein each universal transfer vector contains one unique restriction enzyme cutting site Sal I and allows gene expression cassettes of other items to insert in. The recombinant virus obtained by means of the two universal transfer vectors provided by the invention not only has a growth performance similar to a parent virus, but also has better safety because a plurality of toxicity related genes in a genome are knocked out in an orientation way, and has the potential to be developed into an attenuated vaccine strain for gene engineering.

Description

Technical field [0001] The present invention relates to the preparation of an attenuated vaccine virus vector for animal diseases, and more specifically to the screening method of virus replication non-essential regions that should be mastered in advance for the preparation of recombinant goat pox virus and the construction of a universal transfer vector for mountain pox virus. Technical field. Background technique [0002] Goatpox is an acute, febrile, and contact infectious disease caused by Goatpox virus (GTPV). Sick animals are characterized by fever, systemic acne, respiratory and digestive tract damage, and lymphadenopathy. The outbreak and epidemic of the disease can not only cause huge economic losses to the local sheep breeding industry and the rural economy, but also seriously affect international trade. Therefore, it is listed as a reportable disease by OIE and a type of animal disease by my country. [0003] Vaccination with attenuated virus is an effective means to pr...

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Application Information

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IPC IPC(8): C12N15/09C12N15/10C12N15/63C12N7/01
Inventor 郑敏李春艳刘棋施开创胡杰屈素洁莫胜兰梁媛陆文俊粟艳琼苏凯李军
Owner 广西壮族自治区动物疫病预防控制中心
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