CN114875037B

CN114875037B - Chicken GBP4L gene, expression vector and application

Info

Publication number: CN114875037B
Application number: CN202210612622.4A
Authority: CN
Inventors: 康相涛; 郭玉洁; 田亚东; 闫峰宾; 李国喜; 蒋瑞瑞; 韩瑞丽; 孙桂荣; 李文婷; 李东华; 张彦华; 李转见; 李红; 刘小军
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2023-06-06
Anticipated expiration: 2042-05-31
Also published as: CN114875037A

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a chicken GBP4L gene, an expression vector and application. The nucleotide sequence of the chicken GBP4L gene is shown as SEQ ID NO: 1. The invention proves that the GBP4L gene is related to chicken heat stress for the first time, and can effectively relieve the phenotypes of inflammatory factor increase, immune organ injury and the like caused by chicken heat stress by regulating and controlling the in vivo high expression of the GBP4L gene. The research result of the invention has good application prospect in the aspects of relieving chicken heat stress and breeding heat-resistant stress chicken varieties.

Description

Chicken GBP4L gene, expression vector and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a chicken GBP4L gene, an expression vector and application.

Background

The heat stress seriously affects the development of animal husbandry, causes great loss to the global breeding industry every year, and the current mainstream livestock and poultry variety cannot effectively dissipate heat due to the lack of sweat glands on the skin surface, is sensitive to the change of the environmental temperature, and the heat stress caused by the overhigh temperature brings adverse effects to the production of livestock and poultry. Under the state of heat stress, the electrolyte balance, the glycolipid metabolism, the antioxidant system and the intestinal health of the organism are negatively affected, so that the livestock and poultry have low emotion, inappetence and reduced organism immunity.

How to relieve heat stress of broiler chickens is a problem which puzzles the breeding industry, and the traditional ways of relieving heat stress such as increasing the daily ration energy concentration or applying additives such as electrolyte, mineral substances, vitamins and Chinese herbal medicine preparations have some limiting factors in application, functions and the like. The research on chicken heat stress related genes is hopeful to fundamentally solve chicken heat stress reaction and improve the economic benefit of poultry cultivation.

The Chinese patent application with publication number of CN101948832A discloses a heat resistance related molecular marker on a heat stress protein 70 gene, which is obviously related to the heat resistance property of chickens, and aims to cultivate chickens of heat-resistant strains through a genetic breeding method. Genetic breeding by using auxiliary molecular markers is one of ways for solving chicken heat stress reaction, and genes related to chicken heat stress are discovered, so that the action mechanism of chicken heat stress reaction can be further and fundamentally researched, and the effectiveness and pertinence for relieving chicken heat stress are improved.

Disclosure of Invention

The invention aims to provide a chicken GBP4L gene, which proves that the GBP4L gene is related to chicken heat stress for the first time.

The second object of the present invention is to provide an expression vector comprising the above-mentioned chicken GBP4L gene.

A third object of the present invention is to provide adeno-associated recombinant viruses produced from the above expression vectors.

The fourth object of the invention is to provide the application of the chicken GBP4L gene, the expression vector and the adeno-associated recombinant virus.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a chicken GBP4L gene has a nucleotide sequence shown in SEQ ID NO: 1.

The invention proves that the GBP4L gene is related to chicken heat stress for the first time, and can effectively relieve the phenotypes of inflammatory factor increase, immune organ injury and the like caused by chicken heat stress by regulating and controlling the in vivo high expression of the GBP4L gene. The research result of the invention has good application prospect in the aspects of relieving chicken heat stress and breeding heat-resistant stress chicken varieties.

An expression vector containing the chicken GBP4L gene.

The expression vector can be used for constructing corresponding transformant, thereby effectively relieving chicken heat stress.

Preferably, the starting vector of the expression vector is an adeno-associated viral vector.

Adeno-associated recombinant viruses produced using the expression vectors described above.

The gland related recombinant virus technology is used, the safety is good, the technology is mature, and the chicken heat stress can be effectively relieved.

The chicken GBP4L gene, the expression vector and the adeno-associated recombinant virus are applied to relieving chicken heat stress.

Preferably, the GBP4L gene is highly expressed, so that the heat stress of the chicken is relieved.

Preferably, the chicken is a fixed-start chicken.

The application of the chicken GBP4L gene in the breeding of heat stress resistant chicken varieties.

Preferably, the chicken is a fixed-start chicken.

Drawings

FIG. 1 shows the nucleotide sequence and corresponding amino acid sequence of chicken GBP4L gene;

FIG. 2 is a prior art tool carrier map;

FIG. 3 is a final vector map of the present invention;

FIG. 4 shows the positive cloning sequence in the present invention;

FIG. 5 is a graph showing AAV viral packaging results according to the present invention; wherein, (a) AAV virus packaging process (200×); (B) Real-time PCR detection results, 293T-PMT602 is a control plasmid PMT602 transfected 293T cell sample, 293T-PSE5343 is a target plasmid PSE5343 transfected 293T cell sample;

FIG. 6 shows the in vivo expression results of recombinant GBP4L adeno-associated viruses of the invention;

FIG. 7 shows the effect of GBP4L on heat stressed chicken immune-related factors according to the invention;

FIG. 8 shows the effect of GBP4L on spleen tissue structure of heat stressed chicken according to the present invention.

Detailed Description

The invention will now be described in more detail with reference to the drawings and examples. The experimental materials involved in the following examples are illustrated below:

test animals: the fixed-start chickens are from the original yang germplasm resource field of Henan agricultural university.

The test reagents are shown in table 1 below:

TABLE 1 test Main reagent

/>

The main instruments are shown in table 2 below:

table 2 main instrument for test

Example 1 Chicken GBP4L Gene

The nucleotide sequence of the chicken GBP4L gene of the embodiment is shown as SEQ ID NO:1, the nucleotide sequence and the corresponding amino acid sequence of the chicken GBP4L gene are shown in figure 1.

Example 2 expression vectors containing the chicken GBP4L Gene

The expression vector containing the chicken GBP4L gene of this example was obtained by ligating the chicken GBP4L gene with an adeno-associated virus vector (AAV). The carrier patterns of the tool carrier and the final carrier used in this example are shown in fig. 2 and 3, respectively.

The GBP4L sequence is synthesized by Shanghai and constructed on the target vector. Screening positive clone and sequencing and verifying, wherein the sequencing result of the positive clone is shown in figure 4 (SEQ ID NO: 2), which shows that pAAV-GBP4L-3FLAG adeno-associated virus recombinant plasmid is successfully constructed.

1. Recombinant AAV virus-out and packaging

The HEK293 cell line was used to produce adeno-associated recombinant viruses, and HEK293 cells were cell lines obtained by transforming human embryonic kidney cells with sheared adenovirus type 5 DNA, which produced virus particles with infectious capacity when the cells were co-transformed with AAV Helper-Free System three plasmids (including plasmids of interest, pAAV-RC and pHelper).

AAV virus-out and packaging steps:

(1) 24h before transfection, spread 2X 10 in 15cm Petri dishes ⁷ HEK293 cells were cultured in DMEM medium containing 10% FBS, 37℃and 5% CO ₂ Culturing in an incubator for 24 hours;

(2) The cell density on the day of transfection reached 70-80%, pHelper plasmid, pCap/rap plasmid and destination plasmid were added in a ratio of 6:5:3 to a 5ml EP tube and mixed well, 500uL serum free DMEM was added and labeled A;

(3) 110uL of 1 XPI transfection reagent was added to another 1.5ml EP tube and the total volume was made up to 500uL with serum free DMEM medium, labeled B (note: the medium was seen to turn orange in color);

(4) Slowly adding the transfection reagent in the B tube into the A tube, and incubating for 5min at room temperature;

(5) In the incubation process, HEK293 cells for packaging AAV are subjected to liquid exchange, and each dish is exchanged into a serum-free DMEM culture medium;

(6) After incubation, the mixture in tube A was added to the cells evenly, the cells were placed in 37℃with 5% CO after gentle shaking of the dish ₂ Culturing in an incubator for 4-8 hours;

(7) The culture medium is changed into a complete culture medium, and the culture is continued for 72 hours, and then the virus collection is prepared.

2. Recombinant pAAV-GBP4L-3FLAG virus purification

The recombinant pAAV-GBP4L-3FLAG virus was purified as follows:

(1) Collecting supernatant in a cell culture dish in a 50mL centrifuge tube, and centrifuging at 4000rpm for 10min;

(2) The supernatant was discarded and the pellet was resuspended by adding 3mL of 1 XPBS per tube;

(3) Blowing off cells in a culture dish by using 5-10 mL PBS, loading the cells into another 50mL centrifuge tube, and centrifuging at 4000rpm for 5-10min;

(4) The PBS was discarded, and 5mL of 1 XPBS was added to each tube to resuspend the pellet;

(5) Placing all the 50mL centrifuge tubes with the resuspended cells into a liquid nitrogen tank, freezing for 10-15min, taking out, placing into a water bath kettle at 37 ℃ for thawing, and repeatedly freezing and thawing for 4 times;

(6) The lysed cells were centrifuged at 4000rpm for 5min and the supernatant was collected in a new centrifuge tube and added to treat the nuclease, notes: after being cracked by a physical method, the centrifuged cells are not easy to adhere to the wall, the whole cells are stuck together to form a jelly state, the operation step needs caution, and the cracked cells are difficult to remove on the side;

(7) About 7-8mL of supernatant was loaded into a new 15mL centrifuge tube;

(8) Preparing discontinuous iodixanol gradie, sequentially adding 17% layer,25% layer, 40% layer and 60% layer of iodixanol into a centrifuge tube, and adding AAV supernatant solution into the uppermost layer of each tube discontinuous iodixanol gradient;

(9) Balancing a centrifuge tube, and centrifuging at 40,000rpm for 2.5 hours;

(10) Carefully remove the centrifuge tube and collect the band of interest.

The virus concentration steps are as follows:

(1) Preparing an ultrafiltration tube, adding 5mL of 1 XPBS to infiltrate a filter membrane, and centrifuging at 4000rpm for 5min;

(2) The collected viruses were diluted ten times with 1 XPBS and filtered into ultrafiltration tubes with a 0.22um filter head;

(3) Centrifuging the ultrafiltration tube at 4000rpm for 10-20min;

(4) Repeating the

steps

2 and 3, and finally concentrating the virus liquid to 0.8mL;

(5) Sucking the concentrated AAV virus into a 1.5mL EP tube, adding glycerol, and the final concentration of glycerol is 10%;

(6) Viruses were split and 10uL removed for titer detection.

3. Recombinant pAAV-GBP4L-3FLAG virus titer assay

Currently, the titer of AAV is determined by quantitative PCR detection of the genomic copy number of AAV vectors to determine the number of viral particles of AAV. The procedure for determining the titer of the recombinant pAAV-GBP4L-3FLAG virus is as follows: standard pAAV Standard plasmid 10 ³ ，10 ⁴ ，10 ⁵ ，10 ⁶ ，10 ⁷ Diluting the

detection sample

1000 and 10000 times, wherein the reaction system is shown in table 3, and the reaction conditions are as follows: denaturation at 95 ℃ for 10min,40 cycles: 95℃for 10s and 60℃for 35s.

TABLE 3Real-time PCR reaction System

In Table 3, WPREs-F: TTACGCTATGTGGATACG;

WPREs-R：GCAAGAACTAACCAGGAT。

as shown in FIG. 5A, the target plasmid PSE5343 is transfected into a 293T cell sample, the obvious Expression of Green Fluorescent Protein (EGFP) is observed under a fluorescence inversion microscope after transfection, and the Real-time PCR detection result shows (FIG. 5B) that the GBP4L gene is obviously over-expressed, which indicates that the construction of the GBP4L gene over-expression plasmid is successful.

The results of adeno-associated virus titer measurements are shown in Table 4.

TABLE 4 adeno-associated Virus titre results

The result of the titer test shows that the pAAV-GBP4L-3FLAG titer is 5.43 ^* 10 ¹¹ vg/mL。

Example 3 application of alleviating Heat stress in chickens and Breeding of chickens

3.1 animal test design and sample preparation

48 healthy fixed-start cock with similar weight for 28 days are selected as study objects, placed in isolated clean chicken houses, and fed in a cage raising mode, and the illumination, temperature and humidity are strictly controlled in the feeding process. The animals were randomly divided into A, B, C, D groups of 12 chickens. Wherein, group A is a control group, group B is a heat stress group, group C is a heat stress+control AAV group (without insertion of GBP4L recombinant fragment AAV, pAAV-3 FLAG), group D is a heat stress+over-expression GBP4L recombinant AAV group (insertion of GBP4L recombinant fragment AAV, pAAV-GBP4L-3 FLAG). Group C administration of cervical subcutaneous purified pAAV-3FLAG virus with a viral load of 2.43 ^* 10 ¹⁰ vg; group D administration neck subcutaneous purified pAAV-GBP4L-3FLAG virus with a viral load of 2.43 ^* 10 ¹⁰ vg. The control group A is fed at normal temperature, the temperature is controlled at 21+/-1 ℃, the heat stress group B, C, D is fed at 32+/-1 ℃, and the treatment is continued for 4 weeks, namely 28 days.

Slaughter experiments were performed at trial 29 d. Fasted measures are taken 12 hours before slaughtering, and water is normally drunk. Weighing the hollow living body, performing neck exsanguination and sacrifice after diethyl ether anesthesia, collecting blood, placing in a coagulation promoting tube, standing at room temperature for 30min, centrifuging at 3500r/min for 15min, separating serum, and freezing and preserving at-20 ℃. Spleen and bursa tissue were dissected and isolated, the organ surface was blotted dry with filter paper, weighed and data recorded. Immune organs were snap frozen in liquid nitrogen and transferred to-80℃for storage.

3.2 determination of the expression level of GBP4L Gene in spleen tissue

Four A, B, C, D groups of 5 biological replicates were randomly selected and total chicken spleen tissue RNA was extracted separately using the RNAiso Plus kit. The expression level of GBP4L gene in spleen tissue is detected by adopting a qRT-PCR method. The GBP4L gene primer sequence is as follows:

F:5’-ACGCCTGGATCTTCACACTG-3’；

R:5’-CAGTCCTCGGCCTCATCTTC-3’。

the qRT-PCR method is as follows: by reverse transcription of the cDNA obtained, using

96 fluorescent quantitative PCR instrument

The PremixEx TaqTM kit was used for qRT-PCR. The reaction system is shown in Table 5.

TABLE 5 qRT-PCR reaction System

qRT-PCR amplification procedure was as follows: the extension was performed at 95℃for 3min,95℃for 30s,60℃for 30s,72℃for 30s, and 72℃for 10min for 35 cycles. Taking chicken GAPDH gene as an internal reference and adopting 2 ^-ΔΔCt The relative expression level of the gene was calculated by the method. Three biological and three technical replicates were set up for each reaction.

3.3 immune related index detection

Detection of IgG, igM, CD3 in serum by enzyme-linked immunosorbent assay (ELISA) kit ⁺ 、CD4 ⁺ Corticosterone, TNF-alpha, IL-1 beta, IL-6, TGF-beta, NF-kB cytokine concentrations. The specific procedures were strictly followed by the instructions for ELISA kit use. The specific operation is as follows:

(1) Sample adding of standard substance: setting a standard substance hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are respectively added into the standard substance hole;

(2) Sample adding: blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same) and sample holes to be tested are respectively arranged. And adding 40 mu L of sample diluent into the sample hole to be detected on the enzyme-labeled coated plate, and then adding 10 mu L of sample to be detected. Adding a sample at the bottom of an ELISA plate hole, slightly shaking and uniformly mixing without touching the hole wall as much as possible;

(3) Adding enzyme: adding 100 mu L of enzyme-labeled reagent into each hole, except for blank holes;

(4) Incubation: sealing with sealing plate membrane, and incubating at 37deg.C for 60min;

(5) Preparing liquid: diluting 20 times of concentrated washing liquid with distilled water for later use;

(6) Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 30s, discarding, repeating the operation for 5 times, and beating to dry;

(7) Color development: 50 mu L of the color-developing agent A and 50 mu L of the color-developing agent B are added into each hole, and the mixture is gently vibrated and mixed uniformly, and developed for 15min at 37 ℃ in a dark place;

(8) And (3) terminating: add 50. Mu.L of stop solution per well to stop the reaction (blue turning yellow immediately at this time);

(9) And (3) measuring: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm in blank Kong Diaoling. The measurement should be performed within 15min after the addition of the stop solution.

3.4, blood Biochemical index detection

The blood Glucose (GLU), triglyceride (TG), total Cholesterol (TC), glutamic pyruvic transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), cholinesterase (CHE), total Protein (TP), globulin (GLB), albumin (ALB), albedo, lactate Dehydrogenase (LDH) levels were measured using a fully automated biochemical analyzer.

3.5 immune organ index determination

The spleen and bursa immune organ indexes of 12 chickens in each group are measured, and the immune organ indexes are calculated according to the formula:

immune organ index (%) = immune organ weight (g)/chicken live weight (g) ×100%.

3.6 preparation of Paraffin tissue sections

And 5 spleen tissues of chickens are randomly collected in A, B, C, D groups, fixed for more than 24 hours through a 4% formaldehyde solution, then washed and dehydrated for 12 hours, paraffin embedded and sliced, spread and pasted, dewaxed, hematoxylin-eosin dyed, washed and differentiated, rinsed and dehydrated, counterstained, dehydrated and transparent, and finally sealed and then sliced. Histological observations of the sections were performed with a system biological microscope.

3.7 statistical analysis

Statistical analysis was performed using SPASS 20.0 software, experimental data were expressed as mean ± standard deviation, single factor analysis of variance and independent sample t-test were used for statistical analysis, P <0.05 was considered statistically significant, P <0.01 was considered extremely significant, and Graphpad Prism 5 was used for graphical drawing.

3.8, results and analysis

3.8.1 recombinant GBP4L adeno-associated virus in vivo expression results

As shown in FIG. 6, the viral load was 2.43 ^* 10 ¹⁰ The heat stress model chicken injected with vg recombinant GBP4L adeno-associated virus is detected by adopting a qRT-PCR method, and the result shows that the expression level of GBP4L gene in spleen tissue of the chicken is obviously higher than that in a control group (P < 0.05), the GBP4L gene in the heat stress group injected with pAAV-GBP4L-3FLAG vector is obviously higher than that in the control group (P < 0.05), and the GBP4L gene is up-regulated by 7 times, thus indicating that the recombinant GBP4L adeno-associated virus is successfully injected in vivo, and the spleen tissue can obviously express the GBP4L gene in a high degree.

3.8.2 Effect of GBP4L on Heat stress chicken immune-related factors

As shown in FIG. 7, CD3 in serum was detected using ELISA kit ⁺ 、CD4 ⁺ IgG, igM, corticosterone, TNF-alpha, IL-1 beta, IL-6, TGF-beta, NF-kB cytokine concentrations, results show that heat stress group serum CD3 ⁺ 、CD4 ⁺ IgG and IgM levels were significantly lower than those of the control group (P < 0.05), and corticosterone and TNF-alpha, IL-1β, IL-6, TGF- β, and NF-kB cytokine concentrations were significantly higher than those of the control group (P < 0.05). CD3 ⁺ And CD4 ⁺ The T cells, the reactive cell immunity level, the IgG and IgM reactive humoral immunity level and the blood corticosterone level can be used as reliable indexes of the poultry in a stress state, so that the heat stress model is successfully constructed.

Serum CD3 of heat stress + control AAV group ⁺ 、CD4 ⁺ IgG, igM, corticosterone, TNF-alpha, IL-1 beta, IL-6, TGF-beta, NF-kB cytokine concentrations were not significantly different from the heat stress group (P > 0.05). Heat stress + overexpression of GBP4L recombinant AAV-group serum CD3 ⁺ IgG levels significantly higher than in the heat stress group (P < 0.05), CD4 ⁺ IgM levels were higher than in the heat stressed group (P > 0.05) and were not significantly different from the control group (P > 0.05); the heat stress + the recombinant AAV group with GBP4L over-expressed serum corticosterone and TNF-alpha, IL-1 beta, IL-6, TGF-beta, NF-kB cytokine concentration is significantly lower than that of the heat stress group (P < 0.05), and the corticosterone and TNF-alpha, IL-6, TGF-beta, NF-kB cytokine concentration is not significantly different from that of the control group (P > 0.05).

From the above results, it was found that injection of recombinant adeno-associated virus GBP4L gene can effectively alleviate chicken serum CD3 caused by thermal stress ⁺ 、CD4 ⁺ The levels of IgG and IgM are reduced, the concentration of corticosterone and TNF-alpha, IL-1 beta, IL-6, TGF-beta and NF-kB cytokines is increased, the inflammatory reaction caused by heat stress is relieved, and the immune state of the organism is improved.

3.8.3 and GBP4L effects on the Biochemical index of blood of Heat stressed chickens

The results of measuring Glucose (GLU), triglyceride (TG), total Cholesterol (TC), glutamic pyruvic transaminase (ALT), aspartic transaminase (AST), alkaline phosphatase (ALP), cholinesterase (CHE), total Protein (TP), globulin (GLB), albumin (ALB), white ball ratio, lactate Dehydrogenase (LDH) levels in blood using a full-automatic biochemical analyzer are shown in table 6.

TABLE 6 influence of GBP4L on the biochemical index of blood of heat stressed chickens

Note that: different letters represent P <0.05, and the same letters represent P > 0.05.

The results in table 6 show that none of the four GLB, ALB, white ball ratio, TP, LDH differences are significant (P > 0.05) for the control group a, the heat stress group B, the heat stress + the control AAV group C, the heat stress + the GBP4L overexpressing recombinant AAV group D; the heat stress group B GLU, ALT, AST, TC level is significantly higher than the control group a (P < 0.05), the CHE, ALP, TG level is significantly lower than the control group a (P < 0.05), the heat stress + GBP4L recombinant AAV group D group CHE, GLU, AST, TC level is significantly lower than the heat stress group B (P < 0.05), the ALT level is lower than the heat stress group B (P > 0.05), the CHE, ALP, TG level is significantly higher than the heat stress group B (P < 0.05), and the CHE, TG, GLU, ALT, AST, ALP, TC level is not significantly different from the control group a (P < 0.05).

From the above results, it can be seen that injection of recombinant adeno-associated virus GBP4L gene can effectively relieve heat stress by increasing GLU, ALT, AST, TC level and decreasing CHE, ALP, TG level of chicken serum.

3.8.4 Effect of GBP4L on Heat stress chicken immune organ index

Spleen is the largest peripheral immune organ of poultry, participates in cellular immunity and humoral immunity of organisms, and bursa of fabricius is the peculiar central immune organ of poultry, which dominates the humoral immunity of organisms. The weight of immune organ can be used to evaluate the immune status of chicken, the greater the relative weight and absolute weight, the stronger the cellular immunity and humoral immunity of the organism. Immune organ index is an important index reflecting the immune function of poultry. Four spleen and bursa organ indices of control group A, heat stress group B, heat stress+control AAV group C, heat stress+over-expressed GBP4L recombinant AAV group D were examined and the results are shown in Table 7.

TABLE 7 Effect of GBP4L on heat stressed chicken immune organ index

The results show that the indexes of spleen and bursa of Fabricius organs of the heat stress group B are lower than those of the control group A (P is more than 0.05), the indexes of spleen and bursa of Fabricius organs of the heat stress + over-expressed GBP4L recombinant AAV group D are higher than those of the heat stress group B (P is more than 0.05), and the indexes of spleen and bursa of Fabricius organs of the four groups are not obvious (P is more than 0.05).

From the results, the spleen and bursa of Fabricius immune organ indexes of the chicken are reduced due to heat stress, and the pAAV-GBP4L-3FLAG vector is injected to improve the spleen and bursa of Fabricius immune organ indexes of the heat-stressed chicken and relieve the heat stress.

3.8.5 Effect of GBP4L on spleen tissue Structure of Heat stressed chickens

To understand the effect of GBP4L on the spleen tissue structure of heat stressed chickens, four groups A, B, C, D were randomly collected in this study, 5 chicken spleen tissues each were fixed with 4% formaldehyde solution for more than 24h, and paraffin tissue sections were prepared. Histological observations of the sections were performed using a system biological microscope, and the results are shown in fig. 8.

The result shows that the spleen tissue envelope structure of the control group A is clear, red and white marrow is mixed and distributed, the white marrow is dispersed and penetrates through the whole spleen and mainly contains small lymphocytes, sheath arteries and a small quantity of lymph nodes, and the red marrow comprises venous sinuses and reticular spleen sinuses containing reticulocytes, macrophages, lymphocytes and erythrocytes, and no obvious abnormality is seen; the spleen tissue of the heat stress group B can see a small amount of lymphocyte necrosis, and the nuclei shrink or disintegrate (black arrow), and other obvious abnormalities are not seen; the heat stress + control AAV group C tissue can see a large number of lymphocyte necrosis, shrinkage or fragmentation of cell nuclei (black arrow), and the interstitial mass can see more eosinophilic homogeneous mass (red arrow), and other obvious abnormalities are not seen; the heat stress and over-expression GBP4L recombinant AAV group D tissue envelope has clear structure, red and white marrow is mixed and distributed, the white marrow is dispersed and penetrated in the whole spleen, and mainly contains small lymphocytes, sheath arteries and a small amount of lymphatic nodules, and the red marrow comprises venous sinuses and reticular spleen sinuses containing reticulocytes, macrophages, lymphocytes and erythrocytes, and no obvious abnormality is seen.

From the results, the spleen tissue structure of the chicken is damaged due to heat stress, lymphocyte necrosis is caused, and the spleen tissue damage caused by the heat stress can be alleviated by injecting pAAV-GBP4L-3FLAG vector.

Based on the experimental results, individuals with high expression GBP4L genes can be bred, and then the heat-resistant stress chicken variety is bred by utilizing a general breeding technology.

<110> Henan agricultural university

<120> chicken GBP4L gene, expression vector and application

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 1881

<212> DNA

<213> Gallus gallus

<221> chicken GBP4L Gene

<400> 1

atggacgctc cggtgctccc gatgccggct ccgctctgcc tggtgaccaa caaggacggc 60

gtgctcgccc tcaaccccgc agcgctggca gtgctgcagg aggtggccca gcctatggtg 120

gtggtggcca tcgctgggcc ctaccgcacc gggaagtcct tcctgatgaa ccggctggca 180

cagaagcgca ctggcttccc gctgggcccc acggtgcagg cagagaccaa gggcatctgg 240

atgtggtgcc tgcagcaccc acgccagcag ggagtgacct tgatgctgct ggacactgag 300

ggcctgggag accccaaaaa gggtgacagc cacaatgacg cctggatctt cacactggcg 360

ctgctgttgt ccagcaccct ggtgtacaac agcataggca ccatcgacca gaaggcactg 420

gagaacctcg agctggtgac acagctgtcg gagctgatcc gggtgcggga cagggacaag 480

gacggcaaca acagcgttga aagagaagat gaggccgagg actgcgagtt cgtgcgctac 540

ttccccagct ttgtgtgggt ggtgagggac ttcacgctgg agctgcgcgt gggcgagcgg 600

cccatcagtg aggatgagta cctggagcag gtgctggaac tcaagcacgg gcatggccgc 660

aaggtgcaga actacaatgg gacgcggctc tgcctccgca actatttccc cacacggaag 720

tgcttcgtgc tgcctccgcc actgggcaca gaggagtgcg ggcgcatgga ggagctgccc 780

gaggctgcac tggccccacg tttcctccaa caggcagccc agttctgcaa ctacgtgctg 840

agctccacca ggctcaagac gctgcctgac ggcagagcac tgacagggcg agccctgtgc 900

atgctgctgc agagctacgt ggaggccatc aacagcgggc atctgccctg cctggaggga 960

gcagcggcag tgatgatggc caacgagaat gcagcggcgg tggcggcagc gctggaggcc 1020

tataccaggg gcatgcgggg gctgtcactg cccacagagc ctgcacagct ctcggccgcg 1080

catggggagc acctgcatga ggcactcatt gtcttccagc gtcgcagctt ccgggatcgt 1140

gaccaggagc accagcggcg cctcatggaa cagatcagca cagagtacag ccacctgcag 1200

gaggagaatg atgaagcatc acggcagcac tgcaaggcac tgctggctga gctggcacag 1260

gcactgcata ccagcctggc ccgtggggcc tacacacagc ccggaggcta ccgcgcctac 1320

gaggccgagc ggcagcggct gctggagggc tatcggcaag cggagggcaa gggccccaag 1380

gccgaggagg tgctgaatga gttcctggcg gagcgtcggg cagaggcaga ggcagtgctg 1440

aaggcagaca atgcactgag cgaggccgag aagcagctgg aggaccagaa gcagcaggcg 1500

cagctcctgg agcagcggca gaaagcgacg gcggagcgcg aacggcagct ggaggcgctg 1560

ctagaggacg agcgcaacag ctacgcgcag aacctgcaag ccctggaggc caagatgcag 1620

gcggaggcgg agagcgcgca gcgggagctg cagcgggcga tggaagcgaa gctacgggag 1680

cagagagagc tgctgcagcg cggcttcaga gagcaggcgg cgctgatgga gcaggagctg 1740

gcggcgctgc ggcgggagag caacaacaac agcaagcagg agctggcggc ctccattttc 1800

gacaccgtgc gcgccgcctg cgacctcgtc agcacgttaa aactgtccaa gctggccaag 1860

tcacggggaa cagcagtgta g 1881

<210> 2

<211> 2874

<212> DNA

<213> artificial sequence

<220>

<221> vector sequence

<222> (1)...(75)

<220>

<221> cleavage site

<222> (76)...(81)

<220>

<221> GBP4L sequence

<222> (88)...(1965)

<220>

<221> 3flag sequence

<222> (1966)...(2037)

<220>

<221> P2A-EGFP sequence

<222> (2047)...(2838)

<220>

<221> cleavage site

<222> (2839)...(2844)

<220>

<221> vector sequence

<222> (2845)...(2874)

<400> 2

ccgtcagatc gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga 60

ccgatccagc ctccgggatc cgccaccatg gacgctccgg tgctcccgat gccggctccg 120

ctctgcctgg tgaccaacaa ggacggcgtg ctcgccctca accccgcagc gctggcagtg 180

ctgcaggagg tggcccagcc tatggtggtg gtggccatcg ctgggcccta ccgcaccggg 240

aagtccttcc tgatgaaccg gctggcacag aagcgcactg gcttcccgct gggccccacg 300

gtgcaggcag agaccaaggg catctggatg tggtgcctgc agcacccacg ccagcaggga 360

gtgaccttga tgctgctgga cactgagggc ctgggagacc ccaaaaaggg tgacagccac 420

aatgacgcct ggatcttcac actggcgctg ctgttgtcca gcaccctggt gtacaacagc 480

ataggcacca tcgaccagaa ggcactggag aacctcgagc tggtgacaca gctgtcggag 540

ctgatccggg tgcgggacag ggacaaggac ggcaacaaca gcgttgaaag agaagatgag 600

gccgaggact gcgagttcgt gcgctacttc cccagctttg tgtgggtggt gagggacttc 660

acgctggagc tgcgcgtggg cgagcggccc atcagtgagg atgagtacct ggagcaggtg 720

ctggaactca agcacgggca tggccgcaag gtgcagaact acaatgggac gcggctctgc 780

ctccgcaact atttccccac acggaagtgc ttcgtgctgc ctccgccact gggcacagag 840

gagtgcgggc gcatggagga gctgcccgag gctgcactgg ccccacgttt cctccaacag 900

gcagcccagt tctgcaacta cgtgctgagc tccaccaggc tcaagacgct gcctgacggc 960

agagcactga cagggcgagc cctgtgcatg ctgctgcaga gctacgtgga ggccatcaac 1020

agcgggcatc tgccctgcct ggagggagca gcggcagtga tgatggccaa cgagaatgca 1080

gcggcggtgg cggcagcgct ggaggcctat accaggggca tgcgggggct gtcactgccc 1140

acagagcctg cacagctctc ggccgcgcat ggggagcacc tgcatgaggc actcattgtc 1200

ttccagcgtc gcagcttccg ggatcgtgac caggagcacc agcggcgcct catggaacag 1260

atcagcacag agtacagcca cctgcaggag gagaatgatg aagcatcacg gcagcactgc 1320

aaggcactgc tggctgagct ggcacaggca ctgcatacca gcctggcccg tggggcctac 1380

acacagcccg gaggctaccg cgcctacgag gccgagcggc agcggctgct ggagggctat 1440

cggcaagcgg agggcaaggg ccccaaggcc gaggaggtgc tgaatgagtt cctggcggag 1500

cgtcgggcag aggcagaggc agtgctgaag gcagacaatg cactgagcga ggccgagaag 1560

cagctggagg accagaagca gcaggcgcag ctcctggagc agcggcagaa agcgacggcg 1620

gagcgcgaac ggcagctgga ggcgctgcta gaggacgagc gcaacagcta cgcgcagaac 1680

ctgcaagccc tggaggccaa gatgcaggcg gaggcggaga gcgcgcagcg ggagctgcag 1740

cgggcgatgg aagcgaagct acgggagcag agagagctgc tgcagcgcgg cttcagagag 1800

caggcggcgc tgatggagca ggagctggcg gcgctgcggc gggagagcaa caacaacagc 1860

aagcaggagc tggcggcctc cattttcgac accgtgcgcg ccgcctgcga cctcgtcagc 1920

acgttaaaac tgtccaagct ggccaagtca cggggaacag cagtggacta caaggatgac 1980

gatgacaagg attacaaaga cgacgatgat aaggactata aggatgatga cgacaaaact 2040

agttccggaa gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag 2100

aaccctggac cttccggaat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 2160

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 2220

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 2280

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 2340

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 2400

caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 2460

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 2520

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 2580

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 2640

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 2700

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 2760

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 2820

gacgagctgt acaagtaaaa gcttatcgat aatcaacctc tggattacaa aatt 2874

<210> 3

<211> 18

<212> DNA

<213> artificial sequence

<221> WPREs-F

<400> 3

ttacgctatg tggatacg 18

<210> 4

<211> 18

<212> DNA

<213> artificial sequence

<221> WPREs-R

<400> 4

gcaagaacta accaggat 18

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<221> F

<400> 5

acgcctggat cttcacactg 20

<210> 6

<211> 20

<212> DNA

<221> R

<213> artificial sequence

<400> 6

cagtcctcgg cctcatcttc 20

Claims

1. Chicken (chicken)GBP4LGene, comprising said chickenGBP4LGene expression vector comprising the sameGBP4LApplication of gene adeno-associated recombinant virus in preparation of chicken heat stress relieving products, characterized in thatGBP4LThe nucleotide sequence of the gene is shown in SEQ ID NO: 1.

2. The use according to claim 1, wherein the starting vector of the expression vector is an adeno-associated viral vector.

3. The use according to claim 1, wherein the expression is highGBP4LThe gene can relieve chicken heat stress.

4. The use according to any one of claims 1 to 3, wherein the chicken is a fixed-start chicken.

5. Chicken (chicken)GBP4LApplication of gene in breeding heat stress resistant chicken varietyCharacterized in that the chickenGBP4LThe nucleotide sequence of the gene is shown in SEQ ID NO: 1.

6. The use according to claim 5, wherein the chicken is a fixed-start chicken.