NL2031234B1

NL2031234B1 - Method for Promoting Duck T Cell Proliferation and Application Thereof

Info

Publication number: NL2031234B1
Application number: NL2031234A
Authority: NL
Inventors: Dai Manman; You Bowen; Liu Yang; Liao Ming; Sun Hui
Original assignee: Inst Animal Health Guangdong Academy Agricultural Sciences; Univ South China Agricult
Priority date: 2021-04-13
Filing date: 2022-03-10
Publication date: 2022-11-23
Also published as: CN113234676A; CN113234676B; NL2031234A

Abstract

Disclosed is a method for promoting duck T cell proliferation and application thereof, and the method for promoting duck T cell proliferation comprises the following steps: isolating dUCk memory PBMC from peripheral blood after inoculating duck with H5N1HPAIV in vivo; and infecting duck memory PBMC with H5N1HPAIV in vitro, and mixing it with uninfected memory PBMC for culture_ In the present invention, H5N1 HPAIV is used to stimulate CFSE labelled duck memory PBMC, and the proliferation of T cells is detected from three aspects: the shape, quantity and CFSE labelling changes of T cells, and meanwhile, the change of cytokine expression after the proliferation of T cells is detected by qPCR. A preparation method to promote the proliferation of duck T cells was established to provide materials for studying the immune response mechanism of duck T cells to H5N1HPAIV.

Description

Method for Promoting Duck T Cell Proliferation and Application Thereof

TECHNICAL FIELD The invention belongs to the field of medical biotechnology, and in particular to a method for promoting duck T cell proliferation and application thereof.

BACKGROUND Influenza A virus belongs to the genus orthomyxoviridae influenza virus. The nucleic acid structure of the virus is segmented single-strand negative-strand RNA, which is mainly transmitted through respiratory tract and faecal mouth. During the outbreak of influenza, H5N1 subtype highly pathogenic avian influenza virus (H5N1 HPAIV) can contact people or other mammals through waterfowl (Tian ef a/., 2015). Since H5N1 HPAIV was first discovered in 1997, more than 50% of people died after infection, which caused serious economic losses to the aquaculture industry and posed a great threat to human and animal health (Evseev ef a/., 2019; Enters For Disease C,1997). At present, the research on H5N1 HPAIV mainly focuses on natural immunity, while the research on adaptive immunity is less. Studying the function of duck memory T cells is essential for the evaluation of influenza vaccine and the preparation of new drugs. At present, the research on T cells mainly focuses on mammals, but there is no report on the method of duck T cell proliferation.

SUMMARY The present invention aims at solving at least one of the above technical problems in the prior art. Therefore, the invention provides a preparation method of duck T cells. A preparation method of duck T cells comprises the following steps: S1: isolating duck memory peripheral blood mononuclear cells (PBMC) from peripheral blood after inoculating Anas platyrhyncha domestica with H5N1 HPAIV in vivo; and S2: infecting duck memory PBMC in step S1 with H5N1 HPAIV in vitro, and mixing it with uninfected memory PBMC for culture. Among them, infected duck memory PBMC as APC (antigen presenting cell) presents virus antigen polypeptide, and uninfected memory PBMC as recipient cell responded to the stimulus. In some embodiments of the invention, the duck memory PBMC is isolated from peripheral blood 25-30 days after inoculation of Anas platyrhyncha domestica with H5N1 HPAIV in vivo. In some preferred embodiments of the present invention, the duck memory PBMC is isolated from peripheral blood 28 days after inoculation of Anas platyrhyncha domestica with H5N1 HPAIV.

In some embodiments of the present invention, the H5N1 HPAIV inoculation of Anas platyrhyncha domestica specifically adopts the method of eye drops and nose drops, and each duck is inoculated with 106 EIDs¢/200 pl.

In some embodiments of the present invention, in step S2, cells are infected with a multiplicity of infection (MOI) of 2 - 5.

In some preferred embodiments of the present invention, in step S2, cells are infected with a MOI of 5.

In some embodiments of the present invention, in step S2, cells are collected 6-12 h after infection.

In some preferred embodiments of the present invention, in step S2, cells are collected 6 h after infection.

The present invention also provides a duck T cell prepared by the above method. The invention also provides the application of the duck T cell in preparing vaccines or medicines.

The invention has the beneficial effects that: the invention establishes a preparation method of duck T cells; H5N1 HPAIV is used to stimulate CFSE labelled duck memory PBMC (peripheral blood mononuclear cells), and the proliferation of T cells is detected from three aspects: the shape, number and CFSE labelling changes of T cells, and the optimal inoculation time point and MOI are established; at the same time, the cytokine expression changes after the proliferation of T cells are detected by qPCR. The invention provides materials for studying the immune response mechanism of duck T cells to H5N1 HPAIV.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the cell viability at different times after H5N1 HPAIV infection.

Fig. 2 shows the flow analysis gating of NP-FITC.

Fig. 3 shows the NP positive rate of HSN1 HPAIV infection at different times analysed by flow cytometry.

Fig. 4 shows the NP positive rate at different times after infection.

Fig. 5 shows the cell viability of PBMC in Anas platyrhyncha domestica inoculated with H5N1 HPAIV with different MOI.

Fig. 6 shows the flow analysis gating of NP FITC-A.

Fig. 7 shows the NP positive rate of PBMC infected by H5N1 HPAIV with different MOI detected by flow cytometry.

Fig. 8 shows the NP positive rate of PBMC in Anas platyrhyncha domestica inoculated with H5N1 HPAIV with different MOI.

Fig. 9 shows the results of PCR amplification of NP gene. Fig. 10 is a comparison result of NP gene after sequencing.

Fig. 11 is an experiment of flow detection of CFSE labelled duck memory PBMC proliferation stimulated by H5N1 HPAIV. In which Duck CFSE PBMC: CFSE labelled sample; 7D H5N1 CFSE PBMC: a sample of H5N1 inoculated with CFSE labelled T cells for 7 days; 8D H5N1 CFSE PBMC: a sample of H5N1 inoculated with CFSE labelled T cells for 8 days; 14D H5N1 CFSE PBMC: a sample of H5N1 inoculated with CFSE labelled T cells for 14 days.

Fig. 12 is an experiment of flow detection of CFSE labelled duck memory PBMC proliferation stimulated by ConA. Duck CFSE PBMC: CFSE labelled sample; 3D ConA CFSE PBMC: a sample of ConA CFSE labelled T cells for 3 days; 4D ConA CFSE PBMC: a sample of ConA CFSE labelled T cells for 4 days; 5D ConA CFSE PBMC: a sample of ConA CFSE labelled T cells for 5 days.

Fig. 13 shows the cell morphology of proliferative T cells after the memory PBMC was stimulated by H5N1 HPAIV.

Fig. 14 shows the cell morphology of proliferative T cells stimulated with ConA as positive control.

Fig. 15 shows the change of the proliferation number of duck T cells.

Fig. 16 shows the flow analysis gating of CD4.

Fig. 17 shows the flow analysis gating of CD8a * T cell.

Fig. 18 shows the change of CD4*T cell ratio after H5N1 HPAIV stimulation detected by flow cytometry.

Fig. 19 shows the change of CD8a”T cell ratio after H5N1 HPAIV stimulation detected by flow cytometry.

Fig. 20 shows the change of T cell ratio after HSN1 HPAIV stimulation detected by flow cytometry.

Fig. 21 shows the change of the absolute number of T cells after HSN1 HPAIV stimulation.

Fig. 22 shows the changes of cytokines in proliferating duck T cells detected by qPCR.

DESCRIPTION OF THE INVENTION In the following, the concept and technical effects of the present invention will be clearly and completely described with examples to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, other embodiments obtained by the technicians in the field without creative efforts are within the scope of the present invention.

The duck memory PBMC used in this experiment was isolated from the peripheral blood of ducks inoculated with H5N1 HPAIV for 4 weeks. Among them, each duck was inoculated with 10°EID, 200 ul by eye drops and nose drops. H5N1 HPAIV is A/Duck/Guangdong/383/2008 (H5N1).

RPMI-1640 medium, FBS Australian foetal bovine serum, 2-mercaptoethanol (2-ME), L- glutamine (100 X), 2-mercaptoethanol (1000 X) and HEPES (100 X) were purchased from GIBCO, USA; Dimethyl sulfoxide (DMSO), saponin (saponin), Breyfus desmosin A(BFA) and sword bean protein (ConA) were purchased from American Sigma Company.

The flow antibodies in the test, such as Anti-chicken NP antibody and Anti-Duck CD8 antibody and CFSE-labelling kit, were purchased from Abcam company, Anti-Duck CD4 antibody was purchased from Southern Biotech company, and Goat Anti-Mouse IgG-FITC was purchased from Abbkine biological company.

Embodiment 1 Establishment of a PBMC model of Anas p/atyrhyncha domestica infected with H5N1 HPAIV in vitro 1) Screening of the best time point for HSN1 HPAIV infection of PBMC in Anas platyrhyncha domestica Three different numbers of memory PBMC of Anas platyrhyncha domestica were selected from liquid nitrogen, put into a constant temperature water bath at 37°C to quickly recover the PBMC in Anas platyrhyncha domestica, and counted with 0.08% trypan blue; 108 cells from each sample were put into a 15 ml reaction tube, centrifuged at 400 g for 5 min, and the cells were suspended in RPMI-1640 medium. H5N1 HPAIV was taken out from the -80°C refrigerator, melted in the 4°C refrigerator, and inoculated with cells with MOI=5; during the experiment, a negative control group without virus was set up, cultured in the 37°C infected cell incubator, and taken out every 15 min to shake gently to avoid cell precipitation. After 1 h, it was washed twice with PBS, 2 ml of RPMI- 1640 medium containing 10% FBS was added, and then it was placed in the incubator of infected cells at 37°C to continue incubation. Cells were collected at 6 h, 12 h, 24 h and 36 h after exposure, the number and viability of cells were counted with 0.08% trypan blue, and NP staining was performed.

The results of cell counting are shown in Fig. 1. From Fig. 1, it can be seen that the cell viability of infected group decreased significantly compared with the control group from 12 h (P <

0.01). However, there is no difference in cell viability between the infected group and the control group at 6 h. Considering the cell viability, the cell state at 8 h is suitable for the follow-up experiment if the experimental results are feasible.

The detection results of flow cytometry are shown in Fig. 2 and Fig. 3, in which Fig. 2 shows the flow analysis gating of NP-FITC. Fig. 3 shows the efficiency of flow cytometry in analyzing NP at different times of H5N1 HPAIV infection. It can be seen that the NP positive rate in H5N1 HPAIV infection was 30.7% after 6 h, 25.1% after 12 h and 46.6% after 24 h, and the highest NP positive rate was 24 h after infection. The statistical results are shown in Fig. 4. From Fig. 4, it can be seen that the difference of NP positive rate between 6 h and 12 h is not significant (P>0.05), but the difference of NP positive rate between 6 h and 24 h is significant (P<0.05). Combined with the analysis of cell viability after infection, the best time for H5N1 HPAIV to infect duck PBMC is 6 h. 2) Screening of the best MOI for HSN1 HPAIV in vitro infection of PBMC in Anas platyrhyncha domestica 5 After resuscitation of PBMC in Anas platyrhyncha domestica, counting with 0.08% trypan blue, H5N1 HPAIV infected 108 cells with MOI=2, 5 and 10, respectively, and the cells were incubated in a cell incubator at 37°C for 1 h. 2 ml PBS was added to the reaction tube, 400 g was centrifuged for 5 min, the cells were washed twice, PBS was discarded, 2 ml RPMI-1840 medium containing 10% FBS was added, and incubated in the cell incubator at 37°C. Cells were collected at 6 h after exposure, the number and viability of cells were counted by 0.08% trypan blue, and then NP protein flow detection was performed.

Among them, the cell counting results are shown in Fig. 5. From Fig. 5, it can be seen that there is no significant difference in cell viability between MOI=2 and MOI=5 (P>0.05), and the cell viability after MOI=2 or MQI=5 infection is higher than that after MOI=10 and MOI=20 infection, with significant difference (P<0.05). Considering the cell viability, the best MOI for virus inoculation is 2 or 5. However, in the follow-up, it is necessary to comprehensively consider the efficiency of cell infection, and select the dose with high positive rate and high cell viability after infection.

The results of flow experiment are shown in Figs. 6 and 7, in which Fig. 6 is the flow analysis gating of NP FITC-A. Fig. 7 shows the NP positive rate of PBMC infected by H5N1 HPAIV with different MOI detected by flow cytometry. It can be seen that the positive rates of NP in the infected experimental group were 76.4%, 82.5%, 83.1% and 89.6%, respectively, compared with the uninfected negative control. Statistical analysis shows that as shown in Fig. 8, it can be seen that there is no statistical difference in NP positive rate among different MOI. Therefore, the best MOI=2 for H5N1 HPAIV inoculation of PBMC in Anas platyrhyncha domestica is 2 or 5, and the NP positive rate of virus inoculation is the highest, and the cell viability can meet the experimental needs.

3) PCR detection of NP gene after H5N1 HPAIVAIV inoculated with PBMC in Anas platyrhyncha domestica at different MOI In the experiment, 105 PBMC were selected and inoculated with H5N1 HPAIV with MOI=2, 5, 10 and 20, respectively. Cells were collected 6 h after infection, and RNA was extracted from 105 PBMC. After reverse transcription, PCR amplification was performed with NP gene primer. The results of agarose gel electrophoresis are shown in Fig. 9, in which band 1-3 are the samples of H5N1 HPAIV infected group, band 4 is the negative control sample without virus, band 5 is a negative contrast with water as template in PCR, and band 6 as a contrast in PCR. Fig. 9 shows that the band size is about 1500 bp, which is consistent with the target gene size. The gel was cut under UV light for gel recovery, and sent to Shenggong Bioengineering Company for sequencing to identify the target gene. Fig. 10 is the comparison result of NP gene after sequencing.

Embodiment 2 T cell proliferation in duck PBMC stimulated by H5N1 HPAIV 1) CFSE detection of peripheral blood lymphocyte proliferation stimulated by H5N1 HPAIV Three different numbers of the memory PBMC in Anas platyrhyncha domestica were selected, the cells were resuscitated, counted by trypan blue, and 107 cells were sucked into a 15 ml centrifuge tube. After 400 g centrifugation, the cells were resuspended with PBS preheated at 37°C, CFSE was diluted with preheated PBS, and 1 ul CFSE stock was added to 5 ml PBS solution, the mixture was fully shaken and mixed. The diluted CFSE solution was quickly transferred to the cell suspension with a pipette gun, and blew and mixed well. Water bath at 37°C for 10 min, centrifugation at 400 g for 5 min, discarding the supernatant, washing with PBS twice, counting the number of cells with trypan blue, suspending the cell precipitation with T cell culture medium to make the concentration 107 cells/ml, taking 5 x 108 cells, and inoculating the cells with H5N1 HPAIV with MOI=2. The remaining labelled cell suspension was placed in a 48-well cell plate with 2 x 108 cells per well, and the T cell medium containing 20 U/ml IL-2 was added to 1 ml. Observe the cells every day, pay attention to the formation of cell colonies and the change of culture medium colour.

CFSE labelled duck PBMC, 108 of which were inoculated with H5N1 HPAIV at MOI=5 for 6 h, were co-cultured with 5x108 labelled memory PBMC without virus, and the cell morphology and culture medium colour were observed every day. After 6 days of co-culture, the cells grew in colonies, and the colour of the medium was light yellow. On the seventh day of culture, the cells were cultured in plates, the cell morphology was observed continuously, and the cells were semi- changed every two days.

The flow results are shown in Figs. 11-12, in which Fig. 11 is an experiment of flow detection of CFSE labelled duck PBMC proliferation stimulated by H5N1 HPAIV. Fig. 12 is an experiment of flow detection of CFSE labelled duck PBMC proliferation stimulated by ConA. It can be seen from Fig. 11 that compared with the initial labelled cells, the cultured cells have obvious proliferation peaks from the seventh day; as can be seen from Fig. 12, compared with the initial labelled cells, when ConA was used as the experimental positive control, the cells had obvious proliferation peaks from the third day of culture.

2) Morphological changes of duck T cell proliferation stimulated by H5N1 HPAIV At different times after the proliferation culture of duck memory PBMC stimulated by H5N1 HPAIV, the results were observed by microscope, as shown in Fig. 13. It can be seen that compared with the cells not stimulated by virus in the control group, the cell culture medium of H5N1 HPAIV stimulated group was light yellow, the cells became bigger and rounder, and the aggregation phenomenon appeared; ConA was used as a positive control to stimulate the proliferation of T cells; at the beginning of the fourth day of culture, the cells were clustered and grew, the results are shown in Fig. 14, while the cells in the control group showed a single scattered growth, and the number of dead cells increased with the increase of culture time. 3) Changes in the number of duck T cell proliferation stimulated by H5N1 HPAIV At different times after the proliferation culture of duck memory PBMC stimulated by H5N1 HPAIV, when the cell morphology changed obviously under microscope, took 10 HI of the well- mixed cell suspension, mixed it with 0.08% trypan blue solution and counted it, and counted the change of the absolute number of cells after H5N1 HPAIV stimulation.

The statistical results are shown in Fig. 15. The results show that compared with the control group, the absolute number of cells in H5N1 HPAIV stimulation group is increasing. 4) Detection of the change of T cell ratio with flow cytometry Seven days after cell culture, 108 cells were counted and stained with Anti-Duck CD8 antibody and Anti-Duck CD4 antibody.

The changes of CD4+T cells and CD8a*T cells were detected by flow cytometry.

In the experiment, 2 x 108 PBMC were inoculated with H5N1 HPAIV with MOI=5, then co- cultured with 107 PBMC at 39°C and 5% CO for 4 h; after 14 days of culture, duck CD4*T and CD8 o*T cells were stained in the cells of the experimental group and the control group.

Fig. 16 shows flow analysis gating of CD4, Fig. 17 shows flow analysis gating of CD8a, Fig. 18 shows the change in the ratio of CD4* T cells detected by flow cytometry after H5N1 HPAIV stimulation, and Fig. 19 shows the change in the ratio of CD8a* T cells detected by flow cytometry after HSN 1 HPAIV stimulation.

The results are statistically analysed.

The results are shown in Fig. 20 and Fig. 21, in which Fig. 20 shows the change of T cell ratio after H5N1 HPAIV stimulation.

Fig. 21 shows the change of the absolute number of T cells after H5N1 HPAIV stimulation.

It can be seen from Fig. 20 that compared with the control group, the ratio of CD4*T cells and CD8a* T cells after H5N1 HPAIV stimulation showed an increasing trend, and the difference was significant (P<0.05). It can be seen from Fig. 21 that the absolute number of CD4*T cells and CD8a* T cells after H5N1 HPAIV stimulation increased compared with the control group. 5) qPCR detection of cytokine expression in proliferating T cells (1) Extraction of cell RNA RNA was extracted according to the instructions of Magen's micro cell RNA extraction kit.

Take a suspension containing 105 cells, centrifuge 400 g for 5 min, discard the supernatant, and gently swirl and shake to loosen the cell clumps.

Add 350 ul Buffer RL to lyse the cells, suck the lysate into a DNA filter column with 2 ml collection tube, centrifuge at 1000 g for 1 min at normal temperature, leave the filtrate, add 70% ethanol of equal volume, gently blow for 5 times with a pipette gun, mix evenly, transfer the mixed solution to an RNA filter column with 2 ml collection tube, centrifuge at 1000 g at normal temperature for 1 min, discard the filtrate, and put the filter column back on the collection tube.

Add 600 ul Buffer RW1, 10,000 g at normal temperature for

1 min, discard the filtrate, add 600 ul Buffer RW2, 10,000 g at normal temperature for 1 min, repeat the previous step, 12000 rpm at normal temperature for 3min, place the filter column in a sterilized 1.5 ml centrifuge tube, add 15 ul of DEPC-treated sterilized water into the center of the filter column, centrifuge at 12,000 r at normal temperature for 1 min, measure the concentration with spectrophotometer, and store the extracted RNA at -80°C. 2) Reverse transcription The reaction system was prepared on ice according to Table 1: Table 1 Reverse transcription system

Instantaneous mixing is uniform, and the reaction is carried out in PCR instrument according to the following procedures: 37°C for 15 min, 85°C for 5 s, and 4°C for storage. (3) Design of fluorescent quantitative PCR primer Using Oligo primer design software to design and synthesize fluorescent quantitative primers, as shown in Table 2. Table 2 Fluorescent quantitative primers of gene in Anas platyrhyncha domestica number F-ATGTTCGTGATGGGTGTGAA(SEQ IN R-CTGTCTTCGTGTGTGGCTGT(SEQ IN al., 2019) F-ACCTGCCTACCTCAGGTGAT(SEQ IN R-CCCCGACATGAGTCCCTTTT(SEQ IN 2011) F-CCAGCCAGCTGTTAGCTCTT(SEQ IN D-Granzyme-K XM027446926 (Adams ef al.

R-GCTGCTGTCAAAACCCACTG(SEQ IN 2009)

R-GGATTTTCAAGCCAGTCAGC(SEQ IN 2013) NO.8) F-ATCAGCTGGCTAAGACCGTG(SEQ IN NO.9) (Wei et al., D-TNF-a NC040075 R-GGGATTGTACAAGGCAGCCA(SEQ IN 2013) NO.10) F-GCCAAGAGCTGACCAACTTC(SEQ IN NO.11) (Cornelissen D-IL-2 AF294323 R-ATCGCCCACACTAAGAGCAT(SEQ IN et al. 2013) NO.12) F-GAAGGAAGAGACTTCATTGCCTTGG (SEQ IN NO.13) (Wei et al., D-MHC-I AB115246 R-CTCTCCTCTCCAGTACGTCCTTCC 2013) (SEQ IN NO.14) F-GGGGAGAGGAAACTGAGAGATG (SEQ IN NO.15) (Cornelissen D-IL-10 JN786941.1 R-TCACTGGAGGGTAAAATGCAGA et al. 2013) (SEQ IN NO.16) F-CCTCAACCAGATCCAGCATT(SEQ IN NO.17) (Liang et al., D-IFN-B AY831397 R-GGATGAGGCTGTGAGAGGAG(SEQ IN 2011) NO.18) F-CCACCTTTACCAGCTTCGAG(SEQ IN NO.19) (Kuchipudi et D-MHC-II AY905539 R-CCGTTCTTCATCCAGGTGAT(SEQ IN al., 2014) NO.20) F-TCCCAGCTTCACAGAACTGC(SEQ IN NO.21) (Adams et al, D-OASL AB618537 R-TACTTGACGAGGCGCAGGAG(SEQ IN 2009) NO.22) F-ATCCTCTCCACGCAGGTTTC(SEQ IN NO.23) D-IL-4 KY427739.1 NCBI R-TGGTGCTCTTTGTCACGATG(SEQ IN NO.24) F-ACTGCTTGAGGGTGGAAATG(SEQ IN NO.25) D-Perforin XM035312928 NCBI R-TGCAGCCATCTTGAGTAGGC(SEQ IN NO.28) F-CCCCCAATCTTCCTTATTTCC{SEQ IN D-IFIT5 NO.27) KX034106.1 NCBI R-TTCCTGTCCTTTCCAACTGC(SEQ IN ww F-TCACACGAAGGCCTATTTTACTGG D-MX1 NM-204609 (Cui of of R-GTCGCCGAAGTCATGAAGGA(SEQ IN 2014) (4) Fluorescence quantitative PCR The concentration of RNA was measured by spectrophotometer, and the extracted RNA was reverse transcribed according to TaKaRa reverse transcription reagent instructions (Cat: RRO36A). According to the fluorescent quantitative primers in Table 2 and GAPDH as internal reference, the results were statistically analysed.

The specific reaction system is shown in Table 3 below: Table 3 Fluorescence quantitative reaction system ema

See Fig. 22 for the expression changes of cytotoxicity-related genes Granzyme A, IL-2, Granzyme K, TNF, IFN-r, Th2 cytokine IL-10 and interferon genes IFN-a and IFN-6. It can be seen that the expression of cytotoxicity-related genes Granzyme A, IL-2, Granzyme K, TNF, IFN-r is significantly up-regulated.

Th2 cytokine IL-10 and interferon genes IFN-a and IFN- were also significantly up-regulated.

Combined with the above results, the ratio and number of CD4*T cells and CD8a*T cells increased, indicating that HSN1 HPAIV stimulation caused significant T cell immune response.

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge range of ordinary technicians in the technical field without departing from the purpose of the present invention.

In addition, embodiments of the present invention and features in the embodiments can be combined with each other without conflict.

SEQUENCE LISTING

<110> South China Agricultural University

<110> Institute of Animal Health, Guangdong Academy of Agricultural Sciences <120> Method for Promoting Duck T Cell Proliferation and Application Thereof <130> SHX-Duck Tcell NL

<150> CN2021190395246.3

<151> 2021-04-13

<160> 30

<170> PatentIn version 3.5

<21e> 1

<211> 20

<212> DNA

<213> Manual sequence

<400> 1 atgttcgtga tgggtgtgaa 20 <2105 2

<211> 20

<212> DNA

<213> Manual sequence

<400> 2 ctgtcttcgt gtgtggctgt 20 <2105 3

<211> 20

<212> DNA

<213> Manual sequence

<400> 3 acctgcctac ctcaggtgat 20 <2105 4

<211> 20

<212> DNA

<213> Manual sequence

<400> 4 ccccgacatg agtccctttt 20 <216> 5

<211> 20

<212> DNA

<213> Manual sequence

<400> 5 ccagccagct gttagctctt 20 <210> 6

<211> 20

<212> DNA

<213> Manual sequence

<400> 6 gctgctgtca aaacccactg 20 <210> 7

<211> 20

<212> DNA

<213> Manual sequence

<400> 7 gctgatggca atcctgtttt 20 <210> 8

<211> 20

<212> DNA

<213> Manual sequence

<400> 8 ggattttcaa gccagtcagc 20 <210> 9

<211> 20

<212> DNA

<213> Manual sequence

<400> 9 atcagctggc taagaccgtg 20 <210> 10

<211> 20

<212> DNA

<213> Manual sequence

<400> 10 gggattgtac aaggcagcca 20 <210> 11

<211> 20

<212> DNA

<213> Manual sequence

<400> 11 gccaagagct gaccaacttc 20 <210> 12

<211> 20

<212> DNA

<213> Manual sequence

<400> 12 atcgcccaca ctaagagcat 20 <210> 13

<211> 25

<212> DNA

<213> Manual sequence

<400> 13 gaaggaagag acttcattgc cttgg 25 <210> 14

<211> 24

<212> DNA

<213> Manual sequence

<400> 14 ctctcctctc cagtacgtcc ttcc 24 <210> 15

<211> 22

<212> DNA

<213> Manual sequence

<400> 15 ggggagagga aactgagaga tg 22 <210> 16

<211> 22

<212> DNA

<213> Manual sequence

<400> 16 tcactggagg gtaaaatgca ga 22 <210> 17

<211> 20

<212> DNA

<213> Manual sequence

<400> 17 cctcaaccag atccagcatt 20 <210> 18

<211> 20

<212> DNA

<213> Manual sequence

<400> 18 ggatgaggct gtgagaggag 20 <210> 19

<211> 20

<212> DNA

<213> Manual sequence

<400> 19 ccacctttac cagcttcgag 20 <210> 20

<211> 20

<212> DNA

<213> Manual sequence

<400> 20 ccgttcttca tccaggtgat 20 <210> 21

<211> 20

<212> DNA

<213> Manual sequence

<400> 21 tcccagcttc acagaactgc 20 <210> 22

<211> 20

<212> DNA

<213> Manual sequence

<400> 22 tacttgacga ggcgcaggag 20 <210> 23

<211> 20

<212> DNA

<213> Manual sequence

<400> 23 atcctctcca cgcaggtttc 20 <210> 24

<211> 20

<212> DNA

<213> Manual sequence

<400> 24 tggtgctctt tgtcacgatg 20 <210> 25

<211> 20

<212> DNA

<213> Manual sequence

<400> 25 actgcttgag ggtggaaatg 20 <210> 26

<211> 20

<212> DNA

<213> Manual sequence

<400> 26 tgcagccatc ttgagtaggc 20 <210> 27

<211> 21

<212> DNA

<213> Manual sequence

<400> 27 cccccaatct tccttatttc c 21 <210> 28

<211> 20

<212> DNA

<213> Manual sequence

<400> 28 ttectgtcct ttccaactgc 20 <210> 29

<211> 24

<212> DNA

<213> Manual sequence

<400> 29 tcacacgaag gcctatttta ctgg 24 <210> 30

<211> 20

<212> DNA

<213> Manual sequence

<400> 30 gtcgccgaag tcatgaagga 20

Claims

CONCLUSIONS

A method for promoting T cell proliferation in ducks, the method comprising the steps of: S1: isolating memory mononuclear peripheral blood cells (PBMC) from peripheral blood of a duck after inoculation of H5N1 HPAIV in the duck; and S2: infect duck memory PBMC in step S1 with H5N1 HPAIV in vitro, and mix with uninfected memory PBMC for further culturing.

The method of claim 1, wherein the duck memory PBMC are isolated from the peripheral blood of a duck inoculated with H5N1 HPAIV for more than 25 days.

The method of claim 1, wherein the cells are infected in step S2 with a multiplicity of infection (MOI) of 2 - 5.

The method of claim 3, wherein the cells are infected in step S2 with a multiplicity of infection (MOI) of 5.

The method of claim 1, wherein the cells are collected in step S2 6 to 12 hours after infection.

The method of claim 1, wherein the cells are collected in step S2 6 hours after infection.

A duck T cell prepared by the method of any one of claims 1-6.

A use of duck T cells according to claim 7 for the preparation of vaccines or medicaments.

A use of the duck T cells according to claim 7 in materials for studying the immune response mechanism of duck T cells to H5N1 HPAIV.

10. An application of H5N1 HPAIV to promote proliferation of duck T cells comprising isolating peripheral blood memory mononuclear blood cells (PBMC) from duck peripheral blood after inoculation of H5N1 HPAIV into Anas platyrhyncha domestica, and infecting duck memory PBMC in step S1 with H5N1 HPAIV in vitro, and mixing with uninfected memory PBMC for further culturing.