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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  • Summary
  • 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 retrovi...

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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