ALV-J (avian leukosis virus-J subgroup) pol geneconserved sequence based siRNA (small interfering ribonucleic acid) recombinant interference vector as well as preparation method and application thereof

A technology of avian leukosis virus and interference carrier, applied in the field of genetic engineering, can solve problems such as accelerated virus mutation and evolution, persistent damage to immune response, unscientific, etc., and achieve the effect of reducing economic losses

Inactive Publication Date: 2014-08-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have confirmed that ALV-J has a lasting damage to the immune response, resulting in low productivity, frequent secondary infections and mixed infections, and J subgroup avian leukemia seriously endangers the healthy development of the aquaculture industry.
In foreign countries, the occurrence of this disease is controlled by purification. Due to the long purification period and high cost, it has not been effectively implemented in my country. We can only carry out large-scale purification by eliminating infected chickens. This method cannot control ALV- J infection, at the same time, unscientific and unreasonable purification measures will accelerate the mutation and evolution of the virus, making the disease more complicated; ALV-J is an enveloped virus, and the viral envelope determines the antigenicity of the virus, and the antigenicity And constantly changing, this has become an insurmountable bottleneck in the development of conventional avian leukosis vaccines

Method used

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  • ALV-J (avian leukosis virus-J subgroup) pol geneconserved sequence based siRNA (small interfering ribonucleic acid) recombinant interference vector as well as preparation method and application thereof
  • ALV-J (avian leukosis virus-J subgroup) pol geneconserved sequence based siRNA (small interfering ribonucleic acid) recombinant interference vector as well as preparation method and application thereof
  • ALV-J (avian leukosis virus-J subgroup) pol geneconserved sequence based siRNA (small interfering ribonucleic acid) recombinant interference vector as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 double-stranded DNA

[0038] Targeting the conserved region of the pol gene, the start site is 4614, the end site is 4633, and the siRNA is designed and synthesized, its base sequence is ACAGATATCAGACAGACTT, and its gene sequence is shown in the sequence table SEQ ID NO: 1;

[0039] Designing and synthesizing the hairpin structure containing the above-mentioned target fragment, wherein the gene sequence of the top strand DNA is shown in the sequence table SEQ ID NO: 2; and the gene sequence of the corresponding bottom strand DNA is shown in the sequence table SEQ ID NO: 3; with ddH 2 O was dissolved to 100 μM, 5-10 μl of each complementary single strand was mixed in pairs, and annealed according to the system given in Table 1. The mixture was heated at 95°C for 5-10 minutes, then left at room temperature for 20 minutes to form double-stranded DNA.

[0040] Table 1. oligo DNA annealing system

[0041] 100μM top strand oligo

Embodiment 2

[0042] The construction of embodiment 2 recombinant interference vector:

[0043] The annealed double-stranded DNA was sterilized with ddH 2 O was further diluted to a concentration of 10 nM, and the system was connected at room temperature for 30-45 minutes according to Table 3.

[0044] Table 2. Enzyme Ligation System

[0045] 5×ligation buffer

Embodiment 3

[0046] Embodiment 3 conversion test

[0047] Take 10 μl of the ligation product to transform 100 μl of competent cells DH5α, spread on LB plates (containing 50 μg / ml spectinomycin), and incubate at 37°C.

[0048] Pick 3 clones from the transformation plate, shake the bacteria to extract the plasmid, and then sequence it to verify whether the sequence of the inserted fragment in the recombinant clone is consistent with the designed oligomeric single-stranded DNA, that is, the sequence of top strand DNA and bottom strand DNA;

[0049] The recombinant vector obtained by cloning was sent to a sequencing company for sequencing. It was found that the recombinant vector was obtained by the present invention. The sequence of the inserted fragment in the recombinant clone was consistent with the designed oligomeric single-stranded DNA sequence. It can be seen that the target fragment has been successfully inserted into the cloning vector. .

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Abstract

The invention relates to the technical field of gene engineering and provides an ALV-J (avian leukosis virus-J subgroup) pol geneconserved sequence based siRNA (small interfering ribonucleic acid) recombinant interference vector. According to the vector, conditions that a pol gene is very conservative and significant to an ALV are used, the gene is used for designing and synthesizing siRNA, a hairpin structure of the siRNA is constructed, after annealingdouble-stranded DNA is further obtained, the annealingdouble-stranded DNA is connected with a vector to construct the recombinant interference vector, theinterference vector and a virus are co-transfected with a cell, finally, the fact that the interference vector can effectively interfere with transcription and replication of an in-vitro cell and an ALV-J in a live chicken body is found, a scientific foundation and technical support are provided for control on avian leukosis-J subgroup, and economic losses caused by ALV-J infection to the poultry industry are reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and provides a siRNA recombinant interference vector based on the conserved sequence of the pol gene of the J subgroup avian leukosis virus and its preparation method and its ability to interfere with the transcription of the J subgroup avian leukosis virus in vitro cells and living chickens with the copied application. Background technique [0002] Subgroup J avian leukemia is a neoplastic disease caused by subgroup J avian leukemia virus (ALV-J). ALV-J is an enveloped retrovirus, and clinical infection in poultry mainly manifests as myelocytoma, immune tolerance, high mortality and growth retardation. From 1997 to 1998, ALV-J broke out globally, causing a devastating blow to the world broiler breeder industry. In addition to myeloma caused by ALV-J infection, erythroblastoma, hemangioma, nephroma, and sarcoma often occur in the late stage of infection. The mortality rate is usuall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/10A61K48/00A61P35/02A61P31/14
Inventor 成子强魏戎蓉
Owner SHANDONG AGRICULTURAL UNIVERSITY
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