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Application of CCND1 in preparation of avian retrovirus production enhancer

A retrovirus and enhancer technology, applied in the field of virology, can solve problems such as accelerated virus evolution and mutation, irrationality, and complex diseases, and achieve the effect of promoting replication and prolonging the S phase time

Active Publication Date: 2021-11-26
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the hyperdenaturation of ALV-J and REV virus envelope proteins and their own immune evasion ability, there is no effective control and prevention of drugs or vaccines, and large groups of purification can only be carried out by eliminating infected chickens, but unreasonable and unscientific purification measures, it will also accelerate the evolution and mutation of the virus, making the disease more complex

Method used

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  • Application of CCND1 in preparation of avian retrovirus production enhancer
  • Application of CCND1 in preparation of avian retrovirus production enhancer
  • Application of CCND1 in preparation of avian retrovirus production enhancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Detection of changes in cell cycle state after avian retrovirus infection of cells

[0037] 1. Cell preparation: DF-1 cells were cultured in 25cm 2 Cell culture flasks, after cell counting, the cell density was adjusted to 0.8×10 8 / well, divided into 3 groups with 3 replicates each:

[0038] In group 1, when the DF-1 cells reached 70% confluence, 1 mL of ALV-J NX0101 virus solution was added, maintained for 2 h, discarded the venom, and added Dulbecco's Modified Eagle Medium (DMEM) medium containing 1% fetal bovine serum, in 5% CO at 37°C 2 The culture was maintained in the incubator for 72 hours, and the cells were collected.

[0039] Group 2, when the DF-1 cells reached 70% confluence, add 1mL REV SNV virus solution, maintain for 2h, discard the venom, add Dulbecco's Modified Eagle Medium (DMEM) medium containing 1% fetal bovine serum, at 37°C 5% CO 2 The culture was maintained in the incubator for 72 hours, and the cells were collected.

[0040] Grou...

Embodiment 2

[0045] Example 2 Proteomic analysis of DF-1 cells infected with ALV-J and DF-1 cells infected with REV

[0046] 1. Cell preparation: add the same cell sample of the same group in Example 1 to the cell lysate RIPA:PMSF (100:1), and harvest the cell protein.

[0047] 2. Protein extraction: After the cells were digested with trypsin, 4 times the volume of lysis buffer (8M urea, 1% protease inhibitor, 3 μM TSA, 50 mM NAM and 2 mM EDTA) were added respectively, and ultrasonically lysed. Centrifuge at 13400×g for 10 min at 4°C to remove cell debris, transfer the supernatant to a new centrifuge tube, and use the BCA kit for protein concentration determination.

[0048] 3. Trypsin hydrolysis: add dithiothreitol to the protein solution to make the final concentration 5mM, and reduce at 56°C for 30min. Afterwards, iodoacetamide was added to make the final concentration 11 mM, and incubated at room temperature in the dark for 15 min. Finally the urea concentration of the sample was dil...

Embodiment 3

[0057] Example 3 Overexpression of CCND1 significantly increases the viral load of ALV-J and REV

[0058] In order to clarify that the activation of CCND1 can promote the replication of ALV-J and REV, this example constructed a CCND1 overexpression plasmid, and verified the promotion effect of CCND1 on the load of ALV-J and REV by overexpressing CCND1.

[0059] 1. Construction of CCND1 overexpression plasmid.

[0060](1) Construction of eukaryotic expression vector. The CCND1 gene is optimized, and the optimized nucleotide sequence is shown in SEQID NO.2. The construction of the eukaryotic expression vector is completed by Gemma Gene Company using the existing technology. The present invention selects pcDNA3.1 eukaryotic expression plasmid, and the Sequence the complete CCND1 gene sequence without mutation and insert it into the plasmid, and then transform the constructed plasmid into the competent cell DH5α. After the transformation is completed, shake the bacteria to extrac...

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Abstract

The invention relates to the field of virology, and particularly provides application of CCND1 in preparation of an avian retrovirus production enhancer; the CCND1 is cyclin D1 (G1 / S-specific cyclin-D1, CCND1); the nucleotide sequence of the CCND1 is as shown in SEQ ID NO.1; the coded amino acid sequence of the CCND1 is as shown in SEQ ID NO.2; the inventor establishes a preparation and use method of the CCND1 as the avian retrovirus production enhancer for the first time; the mechanism of the CCND1 for promoting replication of the avian retrovirus is defined for the first time: the CCND1 regulates and controls the period of a virus host cell to be converted from a G1 period to an S period; and the S period is prolonged, so that replication of the retrovirus is promoted.

Description

technical field [0001] The invention relates to the field of virology, and relates to the application of CCND1 in the preparation of an avian retrovirus production enhancer. Background technique [0002] Retroviruses, also known as retroviruses, belong to a class of RNA viruses, and their genetic information is stored on ribonucleic acid (RNA). The retroviral genome consists of two single-stranded RNAs with a typical retroviral genome structure: 5′-LTR-UTR-gag-pol-env-UTR-LTR-3′, including non-coding regions at both ends ( Uncodingregion, UTR), middle coding region (gag-pol-env), 5' cap structure (m7GpppGmp) and 3' polyadenonucleotide tail (PolyA). [0003] The main clinical manifestations of avian leukosis virus subgroup J (ALV-J) and avian reticuloendotheliosis virus (REV), which are more common among avian retroviruses, are immune tolerance, high mortality, growth retardation, and multiorgan Major features such as tumors appear. ALV-J and REV infections are not only ub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N5/10C12N15/85C12N7/00
CPCC07K14/47C12N15/85C12N7/00C12N2800/22C12N2800/107C12N2740/11051
Inventor 成子强周德方刘晓阳崔熙尧张利赵满达
Owner SHANDONG AGRICULTURAL UNIVERSITY
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