CN114032216B

CN114032216B - New use of double-cortex epinephrine-like kinase 1

Info

Publication number: CN114032216B
Application number: CN202111237322.4A
Authority: CN
Inventors: 成子强; 周静; 周德方; 王桂花; 张利
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2023-07-14
Anticipated expiration: 2041-10-22
Also published as: CN114032216A

Abstract

The invention relates to the field of virology, in particular to a novel application of a dual-cortical adrenergic kinase 1 (DCLK 1), which is used for preparing a subgroup J avian leukosis virus replication enhancer, the inventor finds that the DCLK1 can interact with ALV-J SU protein and promote the transformation of a cell cycle from a G1 phase to a S phase so as to obviously improve the replication amount of the ALV-J, and based on the fact, the inventor further constructs a DCLK1 over-expression plasmid pcDNA3.1-DCLK1 to transfect DF-1 cells, so that the DCLK1 is highly expressed in the cells so as to obviously promote the replication of the ALV-J, the virus yield is improved, and no toxic or side effect is caused on the cells, so that the DCLK1 can be used as the virus replication enhancer for the culture of the ALV-J.

Description

New use of double-cortex epinephrine-like kinase 1

Technical Field

The invention relates to the field of virology, in particular to a novel application of a bicorticoid adrenaline-like kinase 1, which is particularly an application in preparing a J subgroup avian leukosis virus replication enhancer.

Background

The subgroup J avian leukosis virus (ALV-J) is firstly separated from broiler chickens in the United kingdom in 1988, belongs to the genus A retrovirus of the family retrovirus, mainly causes tumors including myeloblastoma, hemangioma, erythroblastoma, sarcoma, nephroma and the like, has a mortality rate of usually 1-5%, can reach 50% in peak period, brings serious economic loss to the poultry industry in China, is mainly prevented and controlled by purification at present, and has not yet been successfully developed.

With the continuous and deep research on ALV-J, the culture quantity of the ALV-J needs to be continuously enlarged, and the virus has typical slow virus characteristics and has a longer replication cycle; and in the state of natural infection of ALV-J, the generated viral load is low, and enough viruses are difficult to obtain for subsequent research, so that the development of the ALV-J research is greatly limited.

Since in a certain volume of static culture, up to 90% of retrovirus is limited by the contact area with the target cells and cannot be infected, most of the related products for increasing viral load exist in the form of virus contact enhancer at present, the principle is that the virus is enriched on the surface of the cells in a large amount through physical action, the contact between the virus and the cells is increased, and the infection of the cells by the virus is promoted to the greatest extent, but the promotion effect on the replication of the virus cannot be ensured because the virus and the physiological process of the cells are not involved.

Therefore, whether a brand new virus replication enhancer of ALV-J can be obtained to improve the yield of the ALV-J, and the problem of low virus yield in production and practice at present is one of the problems which are urgently needed to be solved by the technicians in the field.

Disclosure of Invention

The inventor of the present invention provides a new use of a dual-cortical adrenergic kinase 1 (DCLK 1) aiming at the blank existing in the prior art, the use is to prepare a replication enhancer of avian leukosis virus of subgroup J, the inventor finds that DCLK1 can interact with ALV-J SU protein and promote the transformation of cell cycle from G1 phase to S phase so as to obviously increase the replication of ALV-J, on the basis, the inventor further constructs a DCLK1 over-expression plasmid pcDNA3.1-DCLK1 to transfect DF-1 cells, so that the DCLK1 is highly expressed in the cells so as to obviously promote the replication of ALV-J, the virus yield is improved, and the DCLK1 can be used as a virus replication enhancer for the culture of ALV-J.

The inventors have found that ALV-J belongs to a retrovirus and undergoes processes including adsorption, penetration and uncoating, biosynthesis, assembly and release during replication. Binding of the ALV-J SU protein to its receptor initiates the first step of replication. Upon binding of SU proteins to the receptor, conformational changes of TM proteins are induced, leading to membrane fusion of the virus with the cell, and then the nucleocapsid-bearing viral core enters the cell, starting biosynthesis with the aid of enzymes. The inventors have found for the first time that the ALV-J SU protein can interact with DCLK1 to promote its replication.

In addition, the cell cycle (cell cycle) refers to the whole process of the cell from the completion of one division to the completion of the next division, and is divided into two stages of a cell interval and a cell division period, wherein the interval is divided into three stages of a DNA synthesis early stage (G1 stage), a DNA synthesis period (S stage) and a DNA synthesis later stage (G2 stage), and the inventor finds that DCLK1 can promote the transition of the cell from the G1 stage to the S stage after further research, thereby promoting ALV-J replication.

Based on the two theories, the inventor discovers a novel application of the double-cortex adrenergic kinase 1 (DCLK 1), the application is used for preparing the subgroup J avian leukosis virus replication enhancer, and the application fills the blank in the field.

The specific technical scheme of the invention is as follows:

ALV-J belongs to retrovirus, and electron microscopy ultrathin section shows that the outermost layer of the virus particles is lipoid envelope glycoprotein originated from host cell membrane, and the surface of the virus particles is provided with characteristic radial fiber. The glycosylated protein encoded by the viral env gene constitutes the envelope protein of the virus, and comprises two parts, namely a transmembrane glycoprotein subunit (TM) encoded by the gp37 gene and a membrane surface glycoprotein Subunit (SU) encoded by the gp85 gene. SU contains viral receptor determinants and thus can determine the host range and specificity of the virus. ALV-J undergoes reverse transcription during replication, including adsorption, penetration and uncoating, biosynthesis, assembly and release. Binding of the ALV-J SU protein to its receptor initiates the first step of replication. DCLK1 can interact directly with the ALV-J SU protein, promoting the transformation of the cell cycle from G1 phase to S phase and thus the viral replication of ALV-J.

Based on the inventor' S attempts, differential protein bisepinephrine-like kinase-1 (DCLK 1) highly expressed in ALV-J group is screened out for the first time through proteomics detection (Tandem Mass Tag, TMT method) and detected, and it is proved that DCLK1 functions through interaction with SU protein, promotes the transition of cell cycle from G1 phase to S phase, further promotes virus replication of ALV-J, improves ALV-J yield, and has low cytotoxicity. Accordingly, the inventors have found a novel use of DCLK1 and decided to use DCLK1 as an ALV-J replication enhancer to increase ALV-J yield. The specific technical scheme is as follows:

the nucleotide sequence of the coding region of the double-cortical adrenaline-like kinase 1 is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

Based on the result that proteomics DCLK1 is significantly up-regulated in ALV-J, the invention designs and synthesizes DCLK1 qPCR primers as follows:

F：CCCAGGGAGTGAGAACAA；

r: TACCTCCTTTAGCAGTAGCA; the verified result accords with the histology data;

DCLK1 is highly expressed in DF-1 infected with ALV-J NX0101 strain; then, an over-expression plasmid pcDNA3.1-DCLK1 of DCLK1 and a control plasmid pcDNA3.1-NC are constructed, interference plasmids shDCLK1-1, shDCLK1-2 and a control plasmid sh-NC are respectively transfected into DF-1 infected by an ALV-J NX0101 strain and detected, and the gene transcription and protein expression level prove that the DCLK1 is activated by the ALV-J and promotes virus replication;

laser confocal experiments prove that green fluorescence and red fluorescence are overlapped under a confocal microscope by FITC-labeled gp85 protein and CY 3-labeled DCLK1, and the interaction between the DCLK1 and the ALV-J protein is initially proved;

through co-immunoprecipitation, it is proved that DCLK1 and ALV-J SU protein can directly interact;

flow cytometry proves that over-expression of DCLK1 can promote the transformation of the cell cycle from G1 phase to S phase, so as to promote the virus replication of ALV-J, and the interference is the opposite;

taken together, DCLK1 can be used as an ALV-J replication enhancer to increase ALV-J yield.

More specific steps of the above process are as follows:

1. DCLK1 protein expression level is significantly up-regulated in proteomics infected with ALV-J (fold change > 1.3);

ALV-J activates expression of DCLK 1:

proteomic detection of normal DF-1 cells and DF-1 cells infected with ALV-J virus by TMT (Tandem Mass Tag) was performed 3 replicates per group, and 95 differences in proteomic data were observed between normal and ALV-J infected groupsThe protein is subjected to bioinformatics analysis and related documents are consulted, key up-regulated differential protein DCLK1 closely related to ALV-J virus replication is screened out, and DCLK1 is subjected to fluorescent quantitative PCR primer design and antibody purchase; inoculating ALV-J (TCID) at DF-1 density of about 80% ₅₀ 10 ^-4 )

Cells

24h, 48h and 72h after ALV-J inoculation were collected to extract cellular RNA and total protein, respectively, and the transcription level and protein level of DCLKI and ALV-J were detected by means of qPCR and Western blot. The results show that: DCLK1 is upregulated after infection with ALV-J; ALV-J was demonstrated to activate expression of DCLK 1.

2. DCLK1 interacts with the ALV-J SU protein:

ALV-J infected DF-1, maintained with DMEM containing 1% FBS for 72h, laser confocal experiments were performed, FITC labeled gp85 protein, CY3 labeled DCLK1, and overlapping of green fluorescence and red fluorescence was observed under confocal microscopy, demonstrating that DCLK1 co-localizes with ALV-J envelope protein SU in the cytoplasm; the ALV-J SU protein monoclonal antibody is used as a bait for co-immunoprecipitation, DCLK1 is precipitated, and western blot detection is carried out on a precipitated protein sample, so that the DCLK1 and the ALV-J SU protein can directly interact.

3. Overexpression of DCLK1 promotes ALV-J replication;

the eukaryotic expression vector is constructed by using pcDNA3.1 eukaryotic expression plasmid, the DCLK1 base sequence which is completely sequenced and has no mutation is connected into the pcDNA3.1 eukaryotic expression vector, the constructed plasmid is transformed into DH5 alpha competent cells, after the transformation is completed, the bacterium is shaken and the plasmid is extracted, the large gene is sent for sequencing, and the cell transfection is carried out after the sequencing has no error;

after DF-1 transfection of DCLK1 over-expression plasmid for 12h, ALV-J was inoculated and viral load was detected by qPCR and Western blot after 72h maintenance, and the results showed that: compared to the pcDNA3.1 empty vector group, the viral load of ALV-J was significantly up-regulated (more than 3-fold) in cells transfected with the over-expressed DCLK1 plasmid.

4. Interference DCLK1 significantly inhibits replication of ALV-J:

reverse validation of DCLK1 promotion of ALV-J replication was performed using interfering plasmid shRNA. Two DCLK1 interference plasmids are shDCLK1-1 and shDCLK1-2 respectively constructed by Ji Ma company, and the nucleotide sequences are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively. The interfering plasmid transfected cells were inoculated with ALV-J, which resulted in the finding: after interfering with DCLK1, the viral load of ALV-J was significantly down-regulated compared to the transfected empty group.

5. DCLK1 promotes the conversion of the cell cycle from G1 phase to S phase:

DCLK1 over-expression plasmid and interference plasmid were transfected into DF-1 cells, and after 24 hours, ALV-J was infected, and after collecting cells and staining with ethidium bromide (PI), the changes in cell cycle of each group were detected by flow cytometry, and the results showed that: in cells transfected with the over-expressed DCLK1 plasmid, the cell cycle was shifted from G1 to S phase, prolonging the duration of S phase (1.53-fold) compared to the pcdna3.1 empty vector group, whereas knocking down DCLK1 produced the opposite effect.

Based on the technical scheme, compared with the prior art, the invention has the advantages that:

1. the inventor finds that the expression of DCLK1 can be activated after the avian retrovirus ALV-J infects cells, and the protein can be used as an avian retrovirus ALV-J replication enhancer, and the virus load is increased by more than 3 times compared with a control group when the virus is recovered.

2. A novel method for promoting ALV-J replication by using DCLK1 recombinant plasmid is established, and the method can enable DCLK1 to be continuously expressed in cells and promote virus replication efficiently.

3. The mechanism by which DCLK1 promotes ALV-J replication is clarified: DCLK1 promotes replication of ALV-J by modulating the cycle of the viral host cell from G1 phase to S phase and extending the S phase time.

Drawings

FIG. 1 is a volcanic chart of the difference protein obtained by proteomic screening of the significant difference protein DCLK1 closely related to replication of ALV-J virus in example 1 (the abscissa represents the fold difference of the difference protein, and the ordinate represents the P value);

FIG. 2 is a differential protein cluster heat map, wherein 1-3 are 3 parallels of the normal group, and 4-6 are 3 parallels of the ALV-J infected group (vertical represents clustering of samples, lateral represents clustering of proteins, shorter cluster branches represent higher similarity);

FIG. 3 is a graph of GO enrichment analysis;

FIG. 4 is a schematic diagram showing the relationship of activating DCLK1 expression after infection of DF-1 cells by ALV-J virus,

FIG. A is a histogram of ALV-J virus copy number by qPCR for the extraction of cellular RNA from ALV-J infected DF-1 and normal DF-1 in example 2; b is a schematic diagram of dynamic expression of mRNA of DCLK1 in 0-72h after extracting cell RNA and detecting DCLK1 after ALV-J infection; c is a gray scale chart of the expression quantity of DCLK1 and ALV-J gp85 proteins in ALV-J infected cells and normal cells detected by Western blot in the embodiment 2;

FIG. 5 is a schematic diagram showing SU protein interactions of DCLK1 and ALV-J,

panel A shows the use of anti-DCLK 1 rabbit polyclonal antibody and CY3 labeled secondary antibody for DCLK1 in example 3; the ALV-J SU protein uses an ALV-J SU protein monoclonal antibody and a FITC labeled secondary antibody; merge represents the overlapping schematic of DAPI, anti-DCLK 1 and anti-ALV-J in the ALV-J infected group; b is DF-1 cell lysate infected with ALV-J for 72h in example 3 to precipitate DCLK1 with anti-ALV-J SU antibody, and the elution product contains DCLK1 and ALV-J SU schematic diagram detected by Western blot;

figure 6 is a schematic diagram showing the relationship of DCLK1 to effectively increase the ALV-J loading,

a, B and C in the figure are DCLK1, ALV-JmRNA level histogram and protein level gray scale respectively detected by qPCR and Western blot after DF-1 transfection pcDNA3.1-DCLK1 or control plasmid (pcDNA3.1) in example 4 is inoculated with ALV-J and maintained for 72 hours and cellular RNA is extracted; D. e and F are the mRNA level histogram and protein level gray scale of DCLK1, ALV-J respectively detected by qPCR and Western blot after transfection of DF-1 (shDCLK 1-1, shDCLK1-2 or shNC) with shRNA in example 4, and cell infection with ALV-J and cell RNA extraction;

FIG. 7 is a schematic representation of the over-expression of DCLK1 promoting differentiation of ALV-J infected cell cycle from G1 phase to S phase,

FIG. A is a schematic diagram showing the flow assay performed by ALV-J infecting cells and collecting cell samples after the transfection of DF-1 cells with pcDNA3.1-DCLK1 and the control plasmid pcDNA3.1-NC in example 5; b is a histogram of the A graph for convective detection of different cell cycle fractions; c is a schematic flow chart of ALV-J infected cells and collecting cell samples after transfection of DF-1 (shDCLK 1-1, shDCLK1-2 and shNC) with shRNA in example 5; d is a histogram of the C plot for the flow chart for detecting different cell cycle fractions.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. So that the advantages and features of the invention can be more easily understood by those skilled in the art, and the scope of the invention can be more clearly and clearly defined, the experimental methods used in the following examples are all conventional methods or are directly carried out by gene companies unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

EXAMPLE 1 TMT quantitative proteomic screening and analysis of DF-1 cells infected with ALV-J

1. Preparation of cell samples

DF-1 cell culture at 25CM ² In the cell culture flask, the cells were divided into 2 groups of 3 replicates:

when the degree of fusion of group 1, DF-1 cells reaches 70%, 1mL of ALV-J venom is added and maintained for 2 hours, the venom in the cell bottle is discarded, DMEM medium containing 1% FBS is added, and 5% CO is added at 37 DEG C ₂ The cells were maintained in the incubator for 72 hours (earlier experiments showed that ALV-J infection of DF-1 cells for 72 hours could reach peak ALV-J loading);

group 2, DF-1 cell fusion reached 70%, DMEM containing 1% FBS was added, 5% CO at 37 ℃ ₂ Culturing in an incubator for 72 hours.

2. Protein extraction

After cells were digested with pancreatin, 4 volumes of lysis buffer were added, respectively, and sonicated. Cell debris was removed by centrifugation at 12000g for 10min at 4℃and the supernatant was transferred to a new centrifuge tube and assayed for protein concentration using the BCA kit.

3. Pancreatin enzymolysis

Dithiothreitol was added to the protein solution to a final concentration of 5mM and reduced at 56℃for 30min. Then, iodoacetamide was added to a final concentration of 11mM, and incubated at room temperature for 15min in the dark. Finally, the urea concentration of the sample was diluted to below 2M. Pancreatin was added in a mass ratio of 1:50 (pancreatin: protein) and enzymatic hydrolysis was performed overnight at 37 ℃. Then adding pancreatin in a mass ratio of 1:100 (pancreatin: protein), and continuing enzymolysis for 4 hours.

4. TMT markers

The pancreatin-digested peptide fragment was desalted with Strata X C18 (Phenomnex) and lyophilized in vacuo. The peptide was dissolved with 0.5M TEAB and labeled according to the TMT kit instructions. The operation is described briefly as follows: after thawing, the labeled reagent is dissolved by acetonitrile, mixed with the peptide fragment and incubated for 2 hours at room temperature, and the labeled peptide fragment is desalted and freeze-dried in vacuum.

5. HPLC fractionation

The peptide fragment was fractionated by high pH reverse phase HPLC and the column was Agilent 300 extension C18 (5 μm particle size, 4.6mm inner diameter, 250mm long). The operation is as follows: the peptide fragment grading gradient is 8% -32% acetonitrile, pH 9, 60 components are separated in 60min, then peptide fragments are combined into 18 components, and the combined components are subjected to vacuum freeze drying and then are subjected to subsequent operation.

6. Liquid chromatography-mass spectrometry analysis

The peptide fragments were dissolved in liquid chromatography mobile phase A and then separated using an EASY-nLC 1000 ultra high performance liquid system. Mobile phase a was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile.

Setting a liquid phase gradient: 0-26min,7% -25% B;26-34min,25% -38% B;34-37min,38% -80% B;37-40min,80% B, flow rate maintained at 350nL/min.

The peptide fragments were isolated by ultra-high performance liquid chromatography and injected into an NSI ion source for ionization and then analyzed by Orbitrap FusionTM mass spectrometry. The ion source voltage was set to 2.0kV and both the peptide fragment parent ion and its secondary fragments were detected and analyzed using high resolution Orbitrap.

The scanning range of the primary mass spectrum is set to 350-1550m/z, and the scanning resolution is set to 60,000; the range of the secondary mass spectrum scanning is fixed with a starting point of 100m/z, and the Orbitrap scanning resolution is set to 30,000.

The data acquisition mode uses a data dependent scanning (DDA) program, namely, the first 10 peptide fragment parent ions with highest signal intensity are selected to sequentially enter an HCD collision cell after primary scanning, and fragmentation is carried out by using 35% of fragmentation energy, and secondary mass spectrometry analysis is also carried out sequentially. In order to improve the effective utilization of mass spectrometry, automatic Gain Control (AGC) is set to 5E4, a signal threshold is set to 5000ions/s, the maximum injection time is set to 100ms, and the dynamic exclusion time of tandem mass spectrometry scanning is set to 30 seconds to avoid repeated scanning of parent ions.

7. Database search

Secondary mass spectrometry data was retrieved using Maxquant. And (5) searching parameter settings: the database is UniProt Gallus (29480 sequences), a reverse library is added to calculate false positive rate (FDR) caused by random matching, and a common pollution library is added to the database to eliminate the influence of pollution proteins in the identification result; the enzyme cutting mode is set as Trypsin/P; the number of the missed cut sites is set to 2; the minimum length of the peptide fragment is set to 7 amino acid residues; the maximum modification number of the peptide fragment is set to be 5; the mass error tolerance of the primary parent ions of the First search and the Main search is set to 20ppm and 5ppm respectively, and the mass error tolerance of the secondary fragment ions is set to 0.02Da. Cysteine alkylation is set as an immobilization modification, a variable modification is the oxidation of methionine, the acetylation of the N-terminus of the protein. The quantification method was set to TMT-10plex, and FDR for protein identification and PSM identification was set to 1%.

8. Proteomic analysis

GO functional annotation and significance enrichment analysis: GO notes for proteins are divided into 3 broad categories: biological processes, cellular composition, molecular function. The fischer accurate double-ended assay method was used to test differentially expressed proteins against the identified proteins. GO enrichment test p-value values less than 0.05 were considered significant.

And (3) cluster analysis: functional classification information and corresponding enrichment p-value values (based on 1.3-fold differential protein) of the protein groups used are first collected, and then at least one protein group is screened out as significantly enriched (p-value<0.05 A) functional classification. The p-value data matrix obtained by screening is firstly subjected to log ₁₀ And then applying a Z-transform to each function class by the transformed data matrix. Finally, the data set obtained after Z transformation is made by using a hierarchical clustering (Euclidean distance, average connection clustering) methodSingle-sided cluster analysis. The clustering relationship is visualized by using a heat map drawn by the hemmap.

The results were as follows: from the above analysis, the present inventors identified 95 proteins with significant differences in total, and the results were shown in FIG. 1 as a volcanic map of the differential proteins, including 48 significantly up-regulated proteins and 47 significantly down-regulated proteins. The abscissa represents the fold difference of the differential protein, and the ordinate represents the P value; FIG. 2 is a cluster heat map of differential proteins, the inventors found a protein "DCLK1" not reported in the field of poultry among the up-regulated differential proteins infected with ALV-J, and FIG. 3 shows that DCLK1 participates in single cell biological processes for GO functional analysis.

By bioinformatic analysis of proteomics, it was found that high expression of DCLK1 is likely to have a close relationship with ALV-J replication. The DCLK1 is a double-cortex adrenaline-like kinase 1, the nucleotide sequence of the coding region is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

EXAMPLE 2 ALV-J activation of DCLK1 expression after DF-1 cell infection

In order to study the effect of ALV-J infection on DCLK1 expression, the present example constructed DF-1 cell model of ALV-J infection, and detected the expression of ALV-J and DCLK1 at 24, 48 and 72 hours by qPCR and western blot, respectively.

1. Design of fluorescent quantitative primer

According to the DCLK1 sequence disclosed in Genbank accession No. NM_001257257, a DCLK1 protein CDS sequence (272-2461) is obtained, a primer is designed, and is synthesized and verified by a Huada gene, and the primer sequence is as follows:

F-CCCAGGGAGTGAGAACAA-, SEQ ID NO. 5;

R-TACCTCCTTTAGCAGTAGCA-, SEQ ID NO. 6;

2. preparation of cellular material

DF-1 cells are inoculated on a 12-hole cell culture plate according to the specified cell density, normal and ALV-J groups are arranged, and the mixture is uniformly mixed with 37C 5 percent CO ₂ Culturing overnight in an incubator; inoculating ALV-J NX0101 virus to cell culture plate when cell density reaches about 80%, incubating in incubator for 1.5 hr, discarding virus solution, and changing to1% DMEM culture, respectively collecting 24dpi, 48dpi, 72dpi cell samples, and freezing to-80C refrigerator.

3. Cellular RNA extraction

1) The bottom of the centrifuge tube was flicked to loosen the cell pellet, 350. Mu.l of lysis Buffer RL (please add. Beta. -mercaptoethanol before use) was added, and the pellet was repeatedly blown with a pipette until no significant pellet was present in the lysate.

2) All the solutions were transferred to a filter column CS placed in a collection tube, centrifuged at 12000rpm for 2min, and the filtrate was collected.

3) An equal volume of 70% ethanol was added to the filtrate and mixed well using a pipette. The mixture was transferred to the adsorption column CR3 (mounted in the collection tube), centrifuged at 12000rpm for 1min, the waste liquid in the collection tube was poured off, and the adsorption column CR3 was returned to the collection tube.

4) 350. Mu.l deproteinized liquid RW1, 12000rpm was added to the adsorption column CR3, centrifuged for 1min, and the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

5) Preparing a DnaseI working solution: mu.l of the DnaseI stock solution was placed in a new RNase-Free centrifuge tube, 70. Mu.l of RDD solution was added and gently mixed.

6) To the center of the column CR3, 80. Mu.l of DnaseI working solution was added, and the mixture was left at room temperature for 15 minutes.

7) 350. Mu.l deproteinized liquid RW1, 12000rpm was added to the adsorption column CR3, centrifuged for 1min, and the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.

8) 500. Mu.l of a rinse-off liquid RW (ethanol was added before use) was added to the column CR3, and the mixture was allowed to stand at room temperature for 2min at 12000rpm, centrifuged for 1min, and the waste liquid in the collection tube was poured off, and the column CR3 was returned to the collection tube.

9) Repeating step 8).

10 12000rpm, centrifuging for 2min, and pouring out the waste liquid. Placing the adsorption column CR3 at room temperature for several minutes, and thoroughly airing the residual rinsing liquid in the adsorption material;

11 Transferring the CR3 column into a new RNase-Free centrifuge tube, adding 30-10 μl of RNase-Free dd H ₂ O was left at room temperature for 5min at 1200rpm and centrifuged for 2min to obtain an RNA solution.

4. Reverse transcription of RNA into cDNA:

reverse transcription PCR procedure: RNA concentration and purity were measured, and reverse transcription PCR was performed at 37℃in 1min at 85℃for 5s, according to the instructions of PrimeScript RT Master Mix. 2 μl reaction system:

5、Real-time PCR

and amplifying the genes according to primer sequences by adopting a Real-time PCR SYBR Green fluorescent quantitative PCR kit, collecting fluorescent signals after each cycle is finished, and simultaneously detecting the copy number of the GAPDH genes by using a Real-time PCR instrument to correct the detection results of the target genes. The reaction system was 20. Mu.l, the sample was applied on ice, and the Real-time PCR reaction was as follows: 95 ℃ for 30s;95 ℃ for 5s, 60 ℃ for 35s and 34 cycles; 95 ℃ for 15s;60 ℃ for 1min;95℃for 15s. Three replicates were set for each sample, after the fluorescent reaction was completed, the dissolution profile was analyzed to determine the specificity of the PCR reaction and Ct values were recorded for each set of samples. Utilization 2 ^-△△Ct The relative quantitative analysis of ALV-J gag gene and DCLK1 gene expression quantity is carried out by adopting a method, SPSS 17.0 statistical software is adopted for single factor analysis, P<0.05 is a significant difference, P<0.01 is a very significant difference, indicating that the results are statistically significant.

FIG. 4A can illustrate the success of the construction of a DF-1 cell model for ALV-J infection; FIG. 4B can demonstrate that intracellular mRNA expression levels of DCLK1 are significantly higher in ALV-J infected cells than in uninfected cells, i.e., ALV-J can activate DCLK1 expression.

6. Protein concentration detection

The BCA protein quantification kit detects the concentration of protein as follows:

1) Standard substance dilution: with sterilized PBS or dd H ₂ O dilution of BSA standard:

2) BCA working solution preparation: preparing a proper amount of BCA working solution from the reagent A and the reagent B according to the volume ratio of 50:1 according to the number of samples, and fully and uniformly mixing;

3) 200L of well-mixed BCA working solution is added into each standard substance tube and each sample tube;

4) Then 10 mul of diluted standard substance and denatured sample to be detected are sucked and added into the corresponding tube, and fully and uniformly mixed;

5) Incubating in a constant temperature oven at 37 ℃ for 30min, cooling to room temperature or standing at room temperature for 2h;

6) Detecting the absorbance of the solution by using a ultraviolet spectrophotometer at 562nm wavelength;

7) Drawing a standard curve by taking the light absorption value of the standard liquid as an abscissa and the concentration value as an ordinate;

8) The protein concentration of the sample was calculated from the standard curve.

7. Western Blot experiments

To verify the protein expression level, we selected monoclonal antibodies and polyclonal antibodies to the corresponding marker proteins of the virus and protein for Western blotting detection, specifically comprising the following steps:

1) Sample boiling: taking an equal amount (30 mug of protein content) of suspension sample, adding 5 XSDS protein loading buffer solution, and carrying out metal bath denaturation at 100 ℃ for 5min;

2) Preparing SDS-PAGE gel: 10% of separating gel and 5% of concentrated gel, after the gel is naturally solidified, placing the gel into an electrophoresis device, and adding enough 1 XTris-glycine electrophoresis buffer solution into an electrophoresis tank;

3) Sample adding: the amount added per well was 15. Mu.l. Protein Marker loading was 10 μl/well.

4) Electrophoresis: the sample is concentrated in the concentrated gel by using 80v voltage, after the dye front enters the separation gel (generally about 30 min), the voltage is raised to 120v, and the electrophoresis is continued until bromophenol blue reaches the bottom of the separation gel and starts to swim out of the bottom surface of the gel, and the electrophoresis is terminated.

5) Taking glue: carefully prying the glass plate, cutting the concentrated gel and more than the separation gel part, and putting the gel into an electrotransport buffer solution for wetting;

6) Cutting the film: cutting a PVDF film (thickness 0.22 μm) with the same size as the gel, soaking in methanol for about 30s-2min, and then soaking in electrotransport buffer;

7) Transferring: mounting a transfer device, and a sandwich method: namely, the negative electrode clamp-the foam-rubber cushion-the three layers of filter paper-the gel-the PVDF film-the three layers of filter paper-the foam cushion-the positive electrode clamp, no bubbles are generated between the gel and the PVDF film in the installation process, and if bubbles are generated, the bubbles are gently removed by a glass rod. Putting the transfer device into a transfer electrophoresis apparatus, adding a film transfer buffer solution, carrying out constant-current electrophoresis for about 2 hours at 240mA, transferring proteins onto a PVDF film, and finishing the transfer;

8) Closing: taking off PVDF membrane, cutting off a small angle, marking the front and back, sealing 5% skim milk at room temperature by a shaking table for 1.5h or overnight at 4 ℃, rinsing with TBST buffer solution for 5 times and 5min each time;

9) Incubation resistance: incubating the primary antibody at 4 ℃ overnight or a room temperature shaking table for 1.5h, and washing with TBST buffer solution for 3-5 times, each time for 5min;

10 Secondary antibody incubation: secondary antibody (TBST buffer diluted antibody ratio: HRP-labeled goat anti-rabbit 1:1000, HRP-labeled goat anti-mouse 1:1000), shaking table incubation at room temperature for 1h, TBST buffer washing 3-5 times, 5min each time;

11 Developing: PVDF films were placed in a dark room and ECL developed. This process was performed with reference to BeyoECL Plus (hypersensitive ECL chemiluminescent kit).

FIG. 4C can demonstrate that DCLK1 protein expression levels were significantly higher than in the normal group in ALV-J infected cells.

The results were as follows: the present example infects DF-1 cells with ALV-J and detects dynamic expression of ALV-J and DCLK1 by qPCR and western blot. The results showed that the mRNA and protein level expression amount of DCLK1 increased continuously from 24hpi to 72hpi (P < 0.01) after the ALV-J infection (fig. 4A, 4B and 4C). These results indicate that ALV-J is capable of activating the expression of DCLK 1.

Example 3 interaction of DCLK1 with SU protein of ALV-J

To demonstrate whether ALV-J recruits DCLK1 via SU and DCLK1 interactions, the present example performed a laser confocal assay and an immunoprecipitation analysis:

1. SU protein expression localization detection of DCLK1 and ALV-J by laser confocal

1) Cell pretreatment: cells were cultured in laser confocal dishes, the cultured cells were divided into two groups, the first group, when cells reached 70% confluence, infected with ALV-J, maintained for 72h with DMEM medium containing 1% fetal bovine serum; a second group maintained for 72h with DMEM medium containing 1% fetal bovine serum when the cells reached 70% confluency;

2) Washing: the medium in the dish was discarded, 1mL of pre-warmed PBS was added to wash 3 times, and the remaining medium and dead cells were washed;

3) Fixing cells: 1mL of precooled ethanol and acetone mixed solution (2:3) is added into a plate and maintained for 7min;

4) Washing: adding 2mL of PBS for washing for three times, and 5min each time;

5) Closing: adding 1.5mL of 5% skimmed milk powder, and sealing at 37deg.C for 1 hr;

6) Washing: step (4) is the same as that of the step (4);

7) Incubating primary antibodies: separately incubating DCLK1 and ALV-J antibodies, and incubating at 37 ℃ for 1h;

8) Washing: step (4) is the same as that of the step (4);

9) Incubating a secondary antibody: incubating the secondary antibody, adding CY3 into DCLK1, and incubating FITC with ALV-J;

10 Washing: step (4) is the same as that of the step (4);

11 Nuclear staining: labeling nuclei with a nuclei specific dye DAPI, incubating for 5min at 37 ℃;

12 Washing: step (4) is the same as that of the step (4);

13 Confocal microscope observation.

The laser confocal results in fig. 5A show: DCLK1 and ALV-J are both expressed in the cytoplasm, and the fluorescence of both can overlap, indicating that there is an interaction relationship between them.

2. Co-immunoprecipitation detection of SU protein interactions of DCLK1 and ALV-J

1) Preparing a sample: inoculating ALV-J when DF-1 confluence reaches 70%, maintaining for 2h, and maintaining for 72h by replacing with DMEM medium containing 1%; the medium was discarded and washed 3 times with PBS every 1X 10 ⁶ Adding 200 μl of self-contained lysis/equilibration buffer in the kit, performing ice lysis for 20min, centrifuging at 12000rpm at 4deg.C for 10min, separating the supernatant to obtain cellular protein sample, and detecting whether the sample contains target protein, and performing immunoprecipitation test；

2) Incubating the bait antibody: ALV-J monoclonal antibody 1D4 was added to the lysed protein sample and incubated for 1h at 4 ℃;

3) Immunoprecipitation: first, 100. Mu.l of lysis/equilibration buffer was added to the adsorption column and centrifuged at 3000rpm for 1min at room temperature. Discarding the flow-through and then placing the chromatographic column into a new collection tube; adding a cellular protein sample incubated with the ALV-J antibody, centrifuging at 3000rpm for 1min at room temperature, and transferring the adsorption column into a new centrifuge tube; adding 100 μl of washing buffer to the column, centrifuging at 3000rpm for 1min, and transferring the column to a new centrifuge tube; adding 3-5 μl of neutralization buffer into the adsorption column, adding 35 μl of elution buffer, and centrifuging at 3000rpm for 1 time;

4) Western blot analysis samples: the samples of the first group and the treated samples of the second group were subjected to SDS-PAGE and western blot to determine whether the samples contained DCLK1 and ALV-J SU proteins, respectively.

The ability of DCLK1 to precipitate with the ALV-J monoclonal antibody 1D4 as a bait in FIG. 5B further illustrates that DCLK1 may interact with the ALV-J SU protein.

The results were as follows: this example demonstrates that ALV-J recruits DCLK1 through the interaction of SU and DCLK1, contributing to its function, by laser confocal assay and immunoprecipitation analysis.

Example 4 DCLK1 overexpression, construction of an interfering vector and Effect on ALV-J replication

To assess the biological importance of DCLK1 in ALV-J replication, this example constructed DCLK1 over-expression plasmids and interfering plasmids to verify the promoting effect of DCLK1 on ALV-J load.

1. Optimization of DCLK1 base sequence and construction of DCLK1 over-expression vector

The codon optimization and full sequence synthesis of the CDS region of DCLK1 were completed by Huada gene company. The Huada gene company uses internal software to analyze that DCLK1 base sequence replaces rare bases, and the CAI value is better as the CAI value is close to 1; ligating the synthesized DCLK1 base sequence into pcdna3.1 eukaryotic expression vector using NheI (gctag c) and Xho I (CTCGAG) as cleavage sites; transforming the plasmid into competent cells DH 5. Alpha. And screening positive plasmids using solid agar plates containing ampicillin; picking single colonies and amplifying in a large amount in a culture medium containing ampicillin; the plasmids were then extracted for restriction enzyme identification and sequencing comparison.

2. Design and construction of DCLK1 shRNA

The DCLK1 shRNA is designed and constructed by Ji Ma company, and two shRNAs are constructed according to the base sequence of the CDS region of the DCLK 1: the nucleotide sequences of shDCLK1-1 and shDCLK1-2 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.

3. Plasmid transfection

Transfection assays were performed exactly as required by Roche, inc. X-tremeGENE HP DNA Transfection Reagent. The experiments were divided into pcDNA3.1 empty group, pcDNA3.1-DCLK1 group, shDCLK1-2 group, and shNC group, each group having 3 replicates (n=3). Cell transfection plasmid 8h inoculation of ALV-J for 2h after which the venom was discarded, replaced with DMEM medium containing 1% foetal calf serum for a further 72h and the cells were harvested as required by the experiment.

X-tremeGENE HP DNA Transfection Reagent Liposome transfection procedure:

1) Cell culture: cells were cultured in 12-well plates before transfection, ensuring that the cells remained at optimal concentration and status;

2) And (3) reagent rewarming: heating X-tremeGENE HP DNA Transfection Reagent, plasmid and diluent to about 20deg.C, mixing well X-tremeGENE HP DNA Transfection Reagent;

3) Preparing a solution: using Opti-MEM medium as a dilution of plasmid and transfection reagent, 1. Mu.g of plasmid DNA was added to 100. Mu.l of medium and gently mixed. X-tremeGENE HP DNA Transfection Reagent was added directly to the medium containing the diluted plasmid, the ratio of plasmid DNA to transfection reagent was 1:3, in the process, the gun head does not contact the tube wall of the centrifuge tube; the volume of diluent used is more than 100 μl;

4) Incubation: incubating the transfection complex in an environment at about 20 ℃ for 15min;

5) Transfection: the cells taken out of the incubator do not need to discard the original culture medium in the cell culture plate, and the transfection complex is directly dripped into the cell culture plate;

6) ALV-J infected cells: the original culture medium is discarded after 8 hours of transient transfection of the plasmid, PBS is used for washing 3 times, 600mL of ALV-J NX0101 strain venom is added to each well, the venom is discarded after 2 hours of maintenance at 37 ℃, and DMEM culture medium containing 1% fetal bovine serum is added for further maintenance for 72 hours.

4. Cell material collection, cell RNA extraction, reverse transcription of RNA into cDNA, real-time PCR, protein concentration detection, western Blot experiments (same procedure as 2-7 in example 1).

The results were as follows: this example demonstrates that over-expression of DCLK1 (FIG. 6A) significantly enhanced replication of ALV-J at the mRNA and protein level (FIGS. 6B and 6C), and, correspondingly, knocking down DCLK1 (FIG. 6D) inhibited replication of ALV-J at the mRNA and protein level (FIGS. 6E and 6F), demonstrating that DCLK1 can effectively promote viral replication of ALV-J.

EXAMPLE 5 effect of DCLK1 on the DF-1 cell cycle of ALV-J infection

To investigate whether DCLK1 promoted replication of the ALV-J virus by affecting the cell cycle status, this example transfected DCLK1 over-expression plasmid and interfering plasmid into ALV-J infected DF-1 cells to verify the effect of DCLK1 on the cell cycle.

1. Cell sample preparation

Cell culture and X-tremeGENE HP DNA Transfection Reagent liposome transfection procedure were as in example 3, step 3;

2. cell collection

1) Carefully collecting cell culture solution into a centrifuge tube for standby, digesting cells with pancreatin until the cells can be blown down by a pipette or a gun head, adding the cell culture solution collected before, blowing down all adherent cells, gently blowing away the cells, and collecting the cells into the centrifuge tube again;

2) Centrifuging at about 3000rpm for 3-5min, precipitating cells, carefully sucking out the supernatant, and leaving about 50 μl of culture medium to avoid sucking away cells;

3) Approximately 1mL of ice-bath pre-chilled PBS was added, the cells were resuspended, and transferred to a 1.5mL centrifuge tube. The pelleted cells were centrifuged again and the supernatant carefully removed, about 50 μl of PBS could remain to avoid pipetting away. Lightly flicking the bottom of the centrifugal tube to disperse cells properly and avoid cell agglomeration;

3. cell fixation

1) Adding the mixture into 1mL of 70% ethanol precooled in an ice bath, lightly blowing and uniformly mixing, and fixing at 4 ℃ for 12 hours;

2) Centrifuging at about 3000rpm for 3-5min, precipitating cells, carefully pipetting out the supernatant, and leaving about 50 μl of 70% ethanol to avoid pipetting away cells;

3) About 1mL of ice-bath pre-chilled PBS was added to resuspend the cells. The pelleted cells were centrifuged again and the supernatant carefully removed, about 50 μl of PBS could remain to avoid pipetting away. Lightly flicking the bottom of the centrifugal tube to disperse cells properly and avoid cell agglomeration;

4. dyeing

Preparing ethidium bromide staining solution, adding 0.5mL of the ethidium bromide staining solution into each tube of cell sample, slowly and fully suspending cell sediment, carrying out light-shielding warm bath at 37 ℃ for 30min, and storing in a light-shielding way;

5. flow detection and analysis

Red fluorescence was detected with a flow cytometer at an excitation wavelength of 488nm, while light scattering was detected. Cell DNA content analysis and light scattering analysis were performed using Modfit analysis software.

The results were as follows: this example demonstrates that over-expression of DCLK1 can promote the transformation of the cell cycle from G1 phase to S phase and extend the S phase time (fig. 7A and 7B), thereby promoting viral replication of ALV-J, and vice versa (fig. 7C and 7D).

Sequence listing

<110> Shandong agricultural university

<120> New use of double-cortical adrenergic kinase 1

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2187

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

atgtcgtttg gaagagatat ggagctggaa cattttgatg agcgtgataa agctcagcgg 60

tatggccgag ggtcccgggt aaatggtttg cccagcccta cccacagtgc ccactgcagc 120

ttctaccgaa cccgtactct acagaccctg agttctgaga agaaagccaa gaaagttcgt 180

ttctatcgta atggagaccg gtatttcaaa gggattgtat atgccatctc ccctgaccgg 240

tttagatctt ttgaagctct gctggctgat ctgacccgaa ctctgtctga caatgtgaat 300

ctgccccagg gagtgagaac aatctacaca atcgatggct ccaaaaagat ttcctccttg 360

gaccagctag tagaagggga aagctatgta tgtggttcaa tagagccctt caagaagctg 420

gagtacacga agaatgtaaa cccaaactgg tcagtgaatg ttaagacaac ttctacttct 480

cgttcagtgc cgtctcttgc tactgctaaa ggaggtactc cagatacaaa agaaaataag 540

gatttcatta ggcctaaact agtcactatc atcagaagtg gagtgaaacc acggaaggca 600

gtccggattc tgcttaacaa gaagacagca cattcatttg aacaggttct tactgatata 660

actgatgcca tcaagcttga ttcaggagtc gttaaacgct tgtatacact agatggaaaa 720

caggtgatgt gccttcagga tttctttggc gatgatgaca tttttattgc atgtggaccg 780

gaaaagttcc gttaccagga tgatttcttg ctggatgaaa gtgaatgtcg agtggtgaaa 840

tctacttcct ataccaaaat agcttcaagt tcacgaagga gcaccaccaa gagcccaggt 900

ccatcccgac gcagcaagtc tcctgcttct accagctcag tgaatggaac ccctggtagc 960

caactttcta ctccccgctc tgggaagtct ccaagcccat ctcccaccag cccaggaagc 1020

ctacggaaac agaggagctc ccaacacagt ggctcctcta cctcattagc atccaccaaa 1080

gtttgcagct ctatggatga aaatgatgga cctgcagaag aagtgttgga ggaaggtttc 1140

caggttccag catcaatagc agaacgatat aaagttggaa ggactatagg agatggaaat 1200

tttgctattg tgaaggagtg tatagaaaga tcaaccggta gagagtatgc tctgaaaata 1260

atcaaaaaaa gtaaatgtag aggaaaagag cacatgatcc agaatgaggt gtccattttg 1320

agacgagtca agcatcccaa tattgtactt ctgattgagg agatggacat gccaactgag 1380

ttgtaccttg tcatggaact tgtaaaggga ggagatcttt ttgatgctat tacttcgacc 1440

aacaaataca cagagcggga tgccagtggg atgctttaca atctggccag tgccatcaaa 1500

tatcttcaca gcctgaacat cgttcacagg gatatcaagc cggagaacct cctggtatat 1560

gaacaccaag atggaagcaa gtccctgaag ttaggggact ttggcctagc aaccattgtg 1620

gatggacctc tatacactgt ctgtgggacc ccaacatatg tagctccaga aatcattgct 1680

gaaactgggt atggcttgaa agtggacatc tgggcagcgg gcgtgattac ttacatcctg 1740

ctctgtggtt ttcctccatt ccgtggaagt ggagatgacc aagaagtact ttttgatcag 1800

attttgatgg gacaggtgga ttttccatct ccatattggg acaatgtttc tgactctgca 1860

aaggagctta tcacaatgat gcttcaagta gatgtagatc tccgattctc agccttgcaa 1920

gttcttgaac atccatgggt taatgatgat ggccttccag agaatgaaca tcagctatca 1980

gtagctggga agataaagaa gcatttcaac acaggcccta aaccgaacag cacagcagct 2040

ggagtttctg tcatagcact ggaccacggg tttaccatca agagatcagg gtctttggac 2100

tactaccaac aaccaggaat gtattggata agaccaccgc tcttgataag gagaggcagg 2160

ttttccgacg aagacgcaac caggatg 2187

<210> 2

<211> 729

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Met Ser Phe Gly Arg Asp Met Glu Leu Glu His Phe Asp Glu Arg Asp

1 5 10 15

Lys Ala Gln Arg Tyr Gly Arg Gly Ser Arg Val Asn Gly Leu Pro Ser

20 25 30

Pro Thr His Ser Ala His Cys Ser Phe Tyr Arg Thr Arg Thr Leu Gln

35 40 45

Thr Leu Ser Ser Glu Lys Lys Ala Lys Lys Val Arg Phe Tyr Arg Asn

50 55 60

Gly Asp Arg Tyr Phe Lys Gly Ile Val Tyr Ala Ile Ser Pro Asp Arg

65 70 75 80

Phe Arg Ser Phe Glu Ala Leu Leu Ala Asp Leu Thr Arg Thr Leu Ser

85 90 95

Asp Asn Val Asn Leu Pro Gln Gly Val Arg Thr Ile Tyr Thr Ile Asp

100 105 110

Gly Ser Lys Lys Ile Ser Ser Leu Asp Gln Leu Val Glu Gly Glu Ser

115 120 125

Tyr Val Cys Gly Ser Ile Glu Pro Phe Lys Lys Leu Glu Tyr Thr Lys

130 135 140

Asn Val Asn Pro Asn Trp Ser Val Asn Val Lys Thr Thr Ser Thr Ser

145 150 155 160

Arg Ser Val Pro Ser Leu Ala Thr Ala Lys Gly Gly Thr Pro Asp Thr

165 170 175

Lys Glu Asn Lys Asp Phe Ile Arg Pro Lys Leu Val Thr Ile Ile Arg

180 185 190

Ser Gly Val Lys Pro Arg Lys Ala Val Arg Ile Leu Leu Asn Lys Lys

195 200 205

Thr Ala His Ser Phe Glu Gln Val Leu Thr Asp Ile Thr Asp Ala Ile

210 215 220

Lys Leu Asp Ser Gly Val Val Lys Arg Leu Tyr Thr Leu Asp Gly Lys

225 230 235 240

Gln Val Met Cys Leu Gln Asp Phe Phe Gly Asp Asp Asp Ile Phe Ile

245 250 255

Ala Cys Gly Pro Glu Lys Phe Arg Tyr Gln Asp Asp Phe Leu Leu Asp

260 265 270

Glu Ser Glu Cys Arg Val Val Lys Ser Thr Ser Tyr Thr Lys Ile Ala

275 280 285

Ser Ser Ser Arg Arg Ser Thr Thr Lys Ser Pro Gly Pro Ser Arg Arg

290 295 300

Ser Lys Ser Pro Ala Ser Thr Ser Ser Val Asn Gly Thr Pro Gly Ser

305 310 315 320

Gln Leu Ser Thr Pro Arg Ser Gly Lys Ser Pro Ser Pro Ser Pro Thr

325 330 335

Ser Pro Gly Ser Leu Arg Lys Gln Arg Ser Ser Gln His Ser Gly Ser

340 345 350

Ser Thr Ser Leu Ala Ser Thr Lys Val Cys Ser Ser Met Asp Glu Asn

355 360 365

Asp Gly Pro Ala Glu Glu Val Leu Glu Glu Gly Phe Gln Val Pro Ala

370 375 380

Ser Ile Ala Glu Arg Tyr Lys Val Gly Arg Thr Ile Gly Asp Gly Asn

385 390 395 400

Phe Ala Ile Val Lys Glu Cys Ile Glu Arg Ser Thr Gly Arg Glu Tyr

405 410 415

Ala Leu Lys Ile Ile Lys Lys Ser Lys Cys Arg Gly Lys Glu His Met

420 425 430

Ile Gln Asn Glu Val Ser Ile Leu Arg Arg Val Lys His Pro Asn Ile

435 440 445

Val Leu Leu Ile Glu Glu Met Asp Met Pro Thr Glu Leu Tyr Leu Val

450 455 460

Met Glu Leu Val Lys Gly Gly Asp Leu Phe Asp Ala Ile Thr Ser Thr

465 470 475 480

Asn Lys Tyr Thr Glu Arg Asp Ala Ser Gly Met Leu Tyr Asn Leu Ala

485 490 495

Ser Ala Ile Lys Tyr Leu His Ser Leu Asn Ile Val His Arg Asp Ile

500 505 510

Lys Pro Glu Asn Leu Leu Val Tyr Glu His Gln Asp Gly Ser Lys Ser

515 520 525

Leu Lys Leu Gly Asp Phe Gly Leu Ala Thr Ile Val Asp Gly Pro Leu

530 535 540

Tyr Thr Val Cys Gly Thr Pro Thr Tyr Val Ala Pro Glu Ile Leu Ala

545 550 555 560

Glu Thr Gly Tyr Gly Leu Lys Val Asp Ile Trp Ala Ala Gly Val Ile

565 570 575

Thr Tyr Ile Leu Leu Cys Gly Phe Pro Pro Phe Arg Gly Ser Gly Asp

580 585 590

Asp Gln Glu Val Leu Phe Asp Gln Ile Leu Met Gly Gln Val Asp Phe

595 600 605

Pro Ser Pro Tyr Trp Asp Asn Val Ser Asp Ser Ala Lys Glu Leu Ile

610 615 620

Thr Met Met Leu Gln Val Asp Val Asp Leu Arg Phe Ser Ala Leu Gln

625 630 635 640

Val Leu Glu His Pro Trp Val Asn Asp Asp Gly Leu Pro Glu Asn Glu

645 650 655

His Gln Leu Ser Val Ala Gly Lys Ile Lys Lys His Phe Asn Thr Gly

660 665 670

Pro Lys Pro Asn Ser Thr Ala Ala Gly Val Ser Val Ile Ala Leu Asp

675 680 685

His Gly Phe Thr Ile Lys Arg Ser Gly Ser Leu Asp Tyr Tyr Gln Gln

690 695 700

Pro Gly Met Tyr Trp Ile Arg Pro Pro Leu Leu Ile Arg Arg Gly Arg

705 710 715 720

Phe Ser Asp Glu Asp Ala Thr Arg Met

725

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ggcctaaact agtcactatc a 21

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gagtcaagca tcccaatatt g 21

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 5

cccagggagt gagaacaa 18

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 6

tacctccttt agcagtagca 20

Claims

1. Use of a dual corticoid-like kinase 1 for the preparation of an avian leukosis virus replication enhancer of subgroup J, characterized in that: the nucleotide sequence of the coding region of the double-cortex adrenaline-like kinase 1 is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2; the double-cortical adrenoceptor kinase 1 overexpression plasmid is used as the replication enhancer of subgroup J avian leukemia virus.