J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier as well as preparation method and application thereof

A technology of avian leukosis virus and interference carrier, which is applied in the field of genetic engineering and can solve problems such as changes

Inactive Publication Date: 2014-05-21
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At the same time, ALV-J is an enveloped virus. The viral envelope determines the antigenicity of the virus, and the antigenicity is constantly changing. This has become an insurmountable bottleneck in the development of conventional vaccines against avian leukemia.

Method used

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  • J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier as well as preparation method and application thereof
  • J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier as well as preparation method and application thereof
  • J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier 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] Target the conserved region of the env gene, the start site is 6146, the end site is 6165, design and synthesize siRNA, its base sequence is GTACAGCGATGGAATTATT, and its gene sequence is shown in the sequence table SEQ ID NO: 1;

[0039] Design and synthesize 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 treated with sterile 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 genetic engineering, and provides a J avian leukosis virus subgroup env gene conserved sequence-based siRNA (Small Interfering RNA (Ribonucleic Acid)) recombinant interference carrier. The preparation method comprises the following steps: on the basis that the env gene has important meaning for ALV (avian leukosis virus), designing and synthesizing siRNA by the env gene, building to form a hairpin structure of siRNA, further obtaining annealing double-stranded DNA (Deoxyribonucleic Acid), and connecting the double-stranded DNA with the carrier to build the recombinant interference carrier. After the interference carrier and the virus co-transfect cells are adopted, the interference carrier provided by the invention can effectively interfere the transcription and the duplication of the J avian leukosis virus subgroup in the in-vitro cell and the live chicken, the scientific base and the technical support can be provided for preventing and curing the J avian leukosis virus subgroup, and the economic loss caused by the ALV-J infection in the poultry industry can be 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 J subgroup avian leukosis virus env gene and its preparation method and its ability to interfere with J subgroup avian leukosis virus transcription 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 usually 1% to 5%, and th...

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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