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siRNA recombinant interference carrier based on ALV-J gp85 gene conserved region as target sequence, and preparation method of siRNA recombinant interference carrier

A technology of conserved regions and interference vectors, applied in the field of siRNA recombinant interference vectors and its preparation, can solve the problems of undeveloped effective therapeutic or preventive drugs, vaccines, and ALV-J antigenicity easy to change

Inactive Publication Date: 2017-08-18
GUANGXI UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] Since the antigenicity of ALV-J is prone to change, no effective treatment or prevention drugs or vaccines have been developed yet

Method used

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  • siRNA recombinant interference carrier based on ALV-J gp85 gene conserved region as target sequence, and preparation method of siRNA recombinant interference carrier
  • siRNA recombinant interference carrier based on ALV-J gp85 gene conserved region as target sequence, and preparation method of siRNA recombinant interference carrier
  • siRNA recombinant interference carrier based on ALV-J gp85 gene conserved region as target sequence, and preparation method of siRNA recombinant interference carrier

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Experimental program
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Effect test

Embodiment Construction

[0017] 1.1 strain

[0018] Subgroup J avian leukemia virus strain GX13HG03 (preserved in the applicant's laboratory).

[0019] 1.2 Method

[0020] 1.2.1 Design of siRNA sequence

[0021] According to the conserved region of the HPRS103 strain (Z46390.1) and the J subgroup strain HG03gp85 gene on GenBank as the target sequence, the design follows Renylod rules, thermodynamic rules, secondary structure of target mRNA, and the requirements of the carrier pHBAd-U6-RFP For the oligonucleotide sequence siRNA2, EcoRI and BamHI restriction sites are added to the sequence for easy connection to the vector. siRNA2 has the base sequence of SEQ ID NO: 1 in the sequence table (TCCACAGTATCCTCTGAAC);

[0022] 1.2.2 Construction of recombinant adenovirus vector

[0023] 1.2.2.1 Annealing

[0024] Design and synthesize a hairpin structure synthetic shRNA containing the above-mentioned target fragment, wherein the top strand DNA has the base sequence of SEQ ID NO: 2 in the sequence table, ...

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Abstract

The invention discloses a siRNA recombinant interference carrier based on an ALV-J gp85 gene conserved region as a target sequence, and a corresponding preparation method of the siRNA recombinant interference carrier. In the invention, a gp85 gene is used for designing and synthesizing siRNA to build a hairpin-shaped structure forming the siRNA; further an annealed double-chain DNA is obtained, and then is cloned into an adenovirus carrier pHBAd-U6-RFP to build a recombinant adenovirus carrier pAd-gp85-shRNA2; and after sequencing identification is built successfully, recombinant adenovirus carrier is subjected to homologous recombination with framework plasmids in HEK 293 cells. Experiments show that the carrier can effectively inhibit the replication of GX13HG03 virus in DF-1 cells, and can play a certain preventive and therapeutic effect on ALV-J in chickens. Therefore, the invention provides an effective adjunct means for the purification of J subgroup avian leukemia virus, thus providing a new way for AL prevention and control and antiviral research.

Description

technical field [0001] The invention belongs to the technical field of siRNA recombination, and in particular relates to an siRNA recombination interference vector based on the ALV-J gp85 gene conservative region as a target sequence and a preparation method thereof. Background technique [0002] Subgroup J avian leukemia virus (ALV-J) is the most common exogenous avian leukemia virus. ALV-J is seriously infected in domestic broiler chickens, parent breeder chickens and commercial broiler chickens, which has caused huge losses to the chicken industry and seriously jeopardized the development of my country's chicken breeding industry. The disease can be transmitted horizontally or vertically. The existing effective experience abroad is to eliminate ALV virus positive or antibody positive chickens to purify AL and control the spread of AL. However, the purification cycle of this method is long and the cost is high, so it can only be carried out in large-scale breeding compani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K48/00A61K31/7088A61P31/14
CPCA61K31/7088C12N15/86C12N2710/10043
Inventor 王培坤林璐璐秦丽莉杨永立李海娟磨美兰韦天超黄腾韦平
Owner GUANGXI UNIV
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