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New application of doublecortin-like kinase 1

An epinephrine-like kinase technology, applied in the field of virology, can solve the problems of increasing viral contact, low viral load, limiting the progress of ALV-J research, etc., to achieve the effect of promoting replication and prolonging S phase time

Active Publication Date: 2022-02-11
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Subgroup J avian leukemia virus (ALV-J) was first isolated from broiler chickens in the UK in 1988. , sarcoma, nephroma and other tumors, the mortality rate is usually 1%-5%, and the peak period can reach 50%, which brings serious economic losses to my country's poultry industry. Vaccine developed successfully
[0003] With the deepening of research on ALV-J, it is necessary to continuously expand the culture capacity of ALV-J, and the virus has typical lentiviral characteristics and a longer replication cycle; and in the state of natural infection of ALV-J, the virus produced by it The load is low, and it is difficult to obtain enough virus for follow-up research, which greatly limits the progress of ALV-J research
[0004] Since in a certain volume of static culture, as many as 90% of the retroviruses are limited to the contact area with the target cells and cannot infect, so most of the products related to increasing the viral load are in the form of virus contact enhancers The principle is to enrich the virus on the cell surface by physical action, increase the contact between the virus and the cell, and promote the virus to infect the cell to the greatest extent, but because it does not participate in the physiological process of the virus and the cell, it cannot guarantee the promotion of virus replication.
[0005] Therefore whether can obtain a kind of ALV-J brand-new virus replication enhancing agent to improve ALV-J output, solve the low problem of virus yield encountered in production and practice at present and become one of the problems that those skilled in the art urgently need to solve

Method used

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  • New application of doublecortin-like kinase 1
  • New application of doublecortin-like kinase 1
  • New application of doublecortin-like kinase 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 TMT quantitative proteomics screening and analysis of DF-1 cells infected with ALV-J

[0052] 1. Cell sample preparation

[0053] DF-1 cells were cultured at 25CM 2 In the cell culture flask, the cells were divided into 2 groups with 3 replicates in each group:

[0054] In group 1, when the fusion degree of DF-1 cells reaches 70%, add 1 mL of ALV-J venom and maintain for 2 hours, discard the venom in the cell bottle, add DMEM medium containing 1% FBS, and store at 37°C in 5% CO 2 Maintain in the incubator for 72 hours (preliminary experiments showed that DF-1 cells infected with ALV-J can reach the peak of ALV-J load for 72 hours);

[0055] Group 2, when the confluence of DF-1 cells reaches 70%, add DMEM containing 1% FBS, incubate at 37°C in 5% CO 2 The culture was maintained in the incubator for 72 hours.

[0056] 2. Protein extraction

[0057] After the cells were digested with trypsin, 4 times the volume of lysis buffer was added and lysed by sonicati...

Embodiment 2

[0077] Example 2 Activation of DCLK1 expression after ALV-J infects DF-1 cells

[0078] In order to study the effect of ALV-J infection on the expression of DCLK1, a DF-1 cell model infected with ALV-J was constructed in this example, and the expression of ALV-J and DCLK1 at 24, 48 and 72 hours were detected by qPCR and western blot, respectively.

[0079] 1. Design of fluorescent quantitative primers

[0080] According to the DCLK1 sequence published by Genbank accession number NM_001257257, the CDS sequence (272-2461) of the DCLK1 protein was obtained, and primers were designed, which were synthesized and verified by BGI. The primer sequences are as follows:

[0081] F-CCCAGGGAGTGAGAACAA-, shown in SEQ ID NO.5;

[0082] R-TACCTCCTTTAGCAGTAGCA-, shown in SEQ ID NO.6;

[0083] 2. Preparation of cell material

[0084] Inoculate DF-1 cells on a 12-well cell culture plate according to the specified cell density. 2 Cultivate in the incubator overnight; when the cell density re...

Embodiment 3

[0128] Example 3 DCLK1 interacts with SU protein of ALV-J

[0129] In order to clarify whether ALV-J recruits DCLK1 through the interaction of SU and DCLK1, this example carried out laser confocal experiments and co-immunoprecipitation analysis:

[0130] 1. Laser confocal detection of SU protein expression localization of DCLK1 and ALV-J

[0131] 1) Cell pretreatment: Cells were cultured in laser confocal plates, and the cultured cells were divided into two groups. In the first group, when the cells reached 70% confluence, they were infected with ALV-J and treated with DMEM containing 1% fetal bovine serum. The culture medium was maintained for 72 hours; in the second group, when the cells reached 70% confluence, they were replaced with DMEM medium containing 1% fetal bovine serum and maintained for 72 hours;

[0132] 2) Washing: Discard the medium in the plate, add 1 mL of preheated PBS to wash 3 times, and wash the residual medium and dead cells;

[0133] 3) Fix the cells:...

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Abstract

The invention relates to the field of virology, in particular to novel application of doublecortin-like kinase 1 (DCLK1), wherein the application is to prepare a J subgroup avian leukosis virus replication enhancer. According to the invention, it is found that the DCLK1 can interact with ALV-J SU protein and promote a cell cycle to be converted from the G1 phase to the S phase so as to remarkably increase the ALV-J replication amount, and on this basis, the ALV-J replication amount is remarkably increased. a DCLK1 overexpression plasmid pcDNA3.1-DCLK1 transfected DF-1 cell is constructed, so that the DCLK1 is highly expressed in the cell, ALV-J replication is remarkably promoted, the virus yield is improved, the DCLK1 has no toxic or side effect on the cell, and the DCLK1 can be used as a virus replication enhancer to be applied to culture of ALV-J.

Description

technical field [0001] The present invention relates to the field of virology, in particular to a new application of double cortex adrenaline-like kinase 1, specifically the application in the preparation of J subgroup avian leukosis virus replication enhancer. Background technique [0002] Subgroup J avian leukemia virus (ALV-J) was first isolated from broiler chickens in the UK in 1988. , sarcoma, nephroma and other tumors, the mortality rate is usually 1%-5%, and the peak period can reach 50%, which brings serious economic losses to my country's poultry industry. The vaccine was developed successfully. [0003] With the deepening of research on ALV-J, it is necessary to continuously expand the culture capacity of ALV-J, and the virus has typical lentiviral characteristics and a longer replication cycle; and in the state of natural infection of ALV-J, the virus produced by it The load is low, and it is difficult to obtain enough virus for follow-up research, which greatly ...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N9/12C12N15/54C12N15/85C12R1/93
CPCC12N7/00C12N9/12C12N15/85C12Y207/11001C12N2740/11052C12N2800/106Y02A50/30
Inventor 成子强周静周德方王桂花张利
Owner SHANDONG AGRICULTURAL UNIVERSITY
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