Replication enhancer of avian leukosis virus subgroup J

A technology of avian leukosis virus and enhancer, which is applied in the fields of molecular immunology and virology, can solve the problems of non-continuous promotion of virus replication, limited and other problems, and achieve the effect of promoting ALV-J replication

Inactive Publication Date: 2019-09-20
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the above principles, the ability of such products to increase the load of ALV-J is relatively limited and does not continuously promote virus replication

Method used

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  • Replication enhancer of avian leukosis virus subgroup J
  • Replication enhancer of avian leukosis virus subgroup J
  • Replication enhancer of avian leukosis virus subgroup J

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Proteomic analysis of DF-1 cells infected with ALV-J (TMT method)

[0041] 1) Cell preparation: DF-1 cells are cultured at 25cm 2 Cell culture flask, after cell counting, adjust the cell density to 1.0×10 8 / Hole, divided into 2 groups, 3 repeats in each group:

[0042] Group 1, when DF-1 cells reach 70% confluency, add 1ml ALV-J NX0101 virus solution for 2h, discard the venom, add Dulbecco's Modified Eagle Medium (DMEM) medium containing 1% fetal bovine serum, Cultivate in a 5% CO2 incubator at 37°C for 72 hours, add cell lysate RIPA:PMSF (100:1) to harvest cell protein;

[0043] Group 2, when DF-1 cells reach 70% confluency, add DMEM containing 1% fetal bovine serum, and at 37℃, 5% CO 2 Culture in the incubator for 72 hours; add cell lysate RIPA:PMSF (100:1) to harvest cell protein;

[0044] 2) Protein extraction: After the cells are digested with trypsin, 4 times the volume of lysis buffer (8M urea, 1% protease inhibitor, 3μM TSA, 50mM NAM and 2mM EDTA) is added, a...

Embodiment 2

[0055] Example 2 ALV-J activates the expression of CTHRC1:

[0056] In order to further confirm the promotion effect of ALV-J on CTHRC1, this example established an in vitro cell model of ALV-J infection. The changes of CTHRC1 expression after ALV-J infection were detected by qPCR, western blot and ELISA.

[0057] (1) Fluorescence quantitative PCR / qPCR

[0058] Design qPCR specific primers: the sequence of the avian host protein CTHRC1 qPRC primer is F: 5’-ACGCTGGCTTGGTGGA-3’, R: 5’-CAGTTCTTCAATGATGATACGG-3’;

[0059] The primer sequences of the avian internal reference gene GPADH are F: 5'-GAACATCATCCCAGCGTCCA-3', R: 5'-CGGCAGGTCAGGTCAACAAC-3'.

[0060] ALV-J fluorescent quantitative primer sequence is F: 5′-TGCGTGCGTGGTTATTATTTC-3′, R: 5′-AATGGTGAGGTCGCTGACTGT-3′

[0061] The nucleotide sequence is shown in SEQ ID NO. 5-10 in sequence;

[0062] The primers were synthesized by Qingdao Huada Gene Biotechnology Co., Ltd.

[0063] The cells infected with ALV-J for 24h, 48h, 72h and 96h were...

Embodiment 3

[0074] Example 3 Overexpression of CTHRC1 significantly increases the viral load of ALV-J:

[0075] In order to clarify that the activation of CTHRC1 can promote ALV-J replication, this example constructed a CTHRC1 overexpression plasmid, and verified the promotion effect of CTHRC1 on ALV-J load by overexpressing CTHRC1.

[0076] 1. Construction of CTHRC1 overexpression plasmid

[0077] (1) Construct eukaryotic expression vector. The construction of the CTHRC1 eukaryotic expression vector was handed over to Huada Gene Corporation using the existing technology. The present invention selects the pcDNA3.1 eukaryotic expression plasmid, inserts the fully sequenced CTHRC1 gene sequence without mutation into the plasmid, and then inserts the constructed plasmid Transform into competent cells DH5α. After the transformation is completed, the plasmid is extracted by shaking bacteria and sequenced. The sequencing result completely matches the target sequence.

[0078] (2) Then the constructed ...

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Abstract

The invention provides a replication enhancer of avian leukosis virus subgroup J; the replication enhancer of avian leukosis virus subgroup J is collagen triple helix repeat containing 1 which can be used as the virus replication enhancer to directly, significantly and continuously promote ALV-J replication. When the virus is harvested, the viral load amount increases by two or more times as compared with a control group, and the replication enhancer has no toxic or side effects to cells. A CTHRC1 recombinant plasmid is further constructed to promote ALV-J replication, so that the CTHRC1 can be continuously expressed in the cells, ALV-J replication is continuously promoted, and the expression quantity is improved.

Description

Technical field [0001] The present invention relates to the fields of molecular immunology and virology, and specifically relates to a replication enhancer of subgroup J avian leukemia virus. Background technique [0002] ALV-J is a typical tumorigenic retrovirus. Its main clinical manifestation in chickens is myeloid leukemia, and is mostly characterized by growth retardation, high mortality, immune tolerance, and tumors in multiple tissues and organs. The main feature, since ALV-J was first separated in the UK in 1988, it has caused huge losses to the poultry industry in China and even the world. In recent years, the incidence of avian leukemia in subgroup J in China has gradually declined, but in 2018 At the beginning of the year, another typical myeloid tumor-like subgroup J leukemia broke out in six provinces in China. The inventor found that this outbreak of subgroup J avian leukemia was caused by a variant strain of ALV-J. With the continuous spread of ALV-J in the field,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/47C12N7/00C12R1/93
CPCC07K14/47C12N7/00C12N2740/11052
Inventor 成子强庞宇周德方张利
Owner SHANDONG AGRICULTURAL UNIVERSITY
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