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Long-chain non-coding RNA (Ribonucleic Acid) for inhibiting J subtype avian leukosis virus as well as vector and application thereof

A long-chain non-coding, viral technology, applied in viruses/bacteriophages, DNA/RNA fragments, and stably introducing foreign DNA into chromosomes, etc., can solve the problems of no vaccine and effective antiviral drugs, and achieve inhibition of ALV-J Effects of virus infection and replication capacity

Pending Publication Date: 2022-03-22
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, little is known about the pathogenic mechanism of ALV-J, and there are no available vaccines and effective antiviral drugs. The prevention and control of ALV-J mainly adopts strict purification and control procedures

Method used

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  • Long-chain non-coding RNA (Ribonucleic Acid) for inhibiting J subtype avian leukosis virus as well as vector and application thereof
  • Long-chain non-coding RNA (Ribonucleic Acid) for inhibiting J subtype avian leukosis virus as well as vector and application thereof
  • Long-chain non-coding RNA (Ribonucleic Acid) for inhibiting J subtype avian leukosis virus as well as vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Effect of ALV-J infection on the expression level of lnc-LTR5B

[0033] ALV-J infected CEF and DF-1 cells, and the expression level of lnc-LTR5B at different infection time points was determined by fluorescent quantitative PCR. Specific steps are as follows:

[0034] CEF and DF-1 cells were spread in 12-well plates, and when the cell confluency reached 70%, the medium was replaced with serum-free DMEM, and ALV-J virus (JS09GY3 strain) at MOI 0.1 was added to incubate at MOI 0.1, and set uninfected control group. After 2 hours of infection, the medium was replaced with maintenance medium containing 2% fetal bovine serum (containing 1% penicillin / streptomycin).

[0035] After 12, 24, 36 and 48 hours of infection, the total RNA of CEF cells and DF-1 cells was extracted, reverse-transcribed into cDNA, and the expression level of lnc-LTR5B was detected by fluorescent quantitative PCR. The primer sequences were as follows:

[0036] Upstream primer q-lnc-LTR5B-F:

[0037] ...

Embodiment 2

[0042] Amplification of the full-length nucleotide sequence of lnc-LTR5B by RACE method

[0043] (1) RACE technology identifies the 5' and 3' end sequences of lnc-LTR5B

[0044] Depend on RACE 5' / 3'Kit Kit (Takara, #634859) to complete. First, use Reagent was used to extract the total RNA of chicken embryo fibroblast CEF, and then use RNase-free DNase I to remove the genome. Under the action of SMART ScribeReverse Transcriptase (provided by the RACE kit), 1 μg of genome-deleted RNA was reverse-transcribed into 5′- or 3′-RACE products, respectively.

[0045] Then, follow Instructions for the operation of the RACE 5' / 3' Kit kit, using the universal primer UPM and the 5'-end or 3'-end gene-specific primers (gene-specific primer, GSP) for PCR amplification (RACE agarose gel Electropherogram see figure 2 ), cloned and sequenced to obtain the 5' end and 3' end sequences of lnc-LTR5B for later identification of the end sequence of lnc-LTR5B. Wherein, the nucleotide sequen...

Embodiment 3

[0058] lnc-LTR5B overexpression vector construction

[0059] In this example, the full-length sequence of lnc-LTR5B obtained in Example 2 was used to design full-length amplification primers to construct an lnc-LTR5B overexpression plasmid. Specifically include the following steps:

[0060] (1) Total RNA was extracted from chicken embryo fibroblast CEF by using TRIzol method, and after the genome was removed with RNase-free DNase I, it was reverse transcribed into cDNA using PrimeScript RT reagent Kit (Takara, #RR047A).

[0061] (2) Using the cDNA product obtained in step (1) as a template, use a high-fidelity enzyme to amplify the full-length sequence of lnc-LTR5B with homology arms, wherein the primers RT-lnc-LTR5B-F and RT-lnc- The nucleotide sequence of LTR5B-R is as follows:

[0062] RT-lnc-LTR5B-F:

[0063] 5'-ACCCAAGCTGGCTAGCGTTTCTCTTGCTGGCTGCACAG-3' (SEQ ID NO.8)

[0064] RT-lnc-LTR5B-R:

[0065] 5'-GGCTGATCAGCGGGTTTTTTCTTTGAGTTGCAGGTTA-3' (SEQ ID NO.9)

[0066] ...

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Abstract

The invention discloses a long-chain non-coding RNA (Ribonucleic Acid) for inhibiting a J subtype avian leukosis virus as well as a vector and application thereof. The long-chain non-coding RNA is named as lnc-LTR5B, and the nucleotide sequence of the long-chain non-coding RNA is shown as SEQ ID NO. 1. After high expression, the long-chain non-coding RNA provided by the invention can effectively inhibit ALV-J virus infection and replication ability, can be used for preparing vaccines or drugs for preventing or treating subtype J avian leukosis, provides a new target for researching anti-ALV-J virus drugs, and further can be used as a new prevention and treatment strategy and means through related research.

Description

technical field [0001] The invention belongs to the field of livestock and poultry medicine research, in particular to long-chain non-coding RNA for inhibiting J subtype avian leukemia virus, its carrier and application. Background technique [0002] Avian leukosis is an important disease that seriously endangers poultry industry in my country. Its pathogenic viruses can be divided into ALV-A, B, C, D, E, J and K subgroups. Among them, subgroup J avian leukosis virus is still a major threat to the poultry industry. ALV-J virus infection can cause malignant proliferation of hematopoietic cells and immunosuppression in chickens, resulting in decreased egg production of laying hens, slow growth of broiler chickens, and increased mortality rate of chickens. The outbreak of ALV-J will bring huge losses to the poultry industry. In the prior art, little is known about the pathogenic mechanism of ALV-J, and there are no available vaccines and effective antiviral drugs. Strict puri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90A61K31/7088A61P31/14
CPCC12N15/1132C12N15/85C12N15/902A61K31/7088A61P31/14C12N2310/10C12N2800/106Y02A50/30
Inventor 陈世豪赵睿涵吴挺潘诗雨崔恒宓
Owner YANGZHOU UNIV
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