Construction method and application of ANGPTL3 overexpression laying hen fatty liver hemorrhagic syndrome model

Abstract

The invention relates to a construction method and application of an ANGPTL3 overexpression laying hen fatty liver hemorrhagic syndrome model, and the construction method comprises the following steps: (1) respectively integrating a transposon element carrying a chicken ANGPTL3 coding gene and a transposase gene into an adenovirus vector, and obtaining a recombinant adenovirus by using a virus packaging system; (2) punching the chick embryos developed for 4 days (E4) by adopting an equatorial plane windowing method; (3) introducing the recombinant adenovirus into an E4 chick embryo body in a microinjection manner; and (4) hatching to laying hens to prepare the laying hen fatty liver hemorrhagic syndrome model. The animal disease model is constructed on poultry through protein overexpression for the first time, and the laying hen fatty liver hemorrhagic syndrome model does not need to be fed with high-fat feed and has the advantages of being accurate in animal model construction, remarkable in fatty liver characterization and high in efficiency. The method has a great application value in research on the fatty liver hemorrhagic syndrome of laying hens and drug development and screening of poultry.

Description

Technical Field

[0001] The present invention belongs to the technical field of model construction, and particularly relates to a method for constructing a chicken ANGPTL3 overexpression laying hen fatty liver hemorrhage syndrome model and its application. Background Art

[0002] Laying hen fatty liver hemorrhage syndrome is a metabolic disease similar to human non-alcoholic fatty liver that is highly prevalent in the current poultry breeding industry. It is mainly caused by excessive lipid accumulation in the liver, leading to fatty degeneration of the laying hen's liver. In the later stage of the disease, the laying hen will die due to internal bleeding caused by liver enlargement and rupture. The pathological characteristics of laying hens suffering from fatty liver hemorrhage syndrome include fatty degeneration of the liver, liver enlargement, abdominal fat deposition, etc. Since this disease mostly occurs in high-yield laying hens during the peak and late stages of egg production, and has obvious concealment and suddenness, it can cause sudden death of high-yield laying hens, and has now caused huge economic losses to the poultry industry. In addition, due to the suddenness and concealment of laying hen fatty liver hemorrhage syndrome, it is difficult to diagnose the disease before it occurs. Currently, there is no other effective clinical diagnosis method except pathological dissection, resulting in very limited prevention means. At present, there is no effective treatment drug or treatment measure for this disease, and the only solution is to slow down or reduce the prevalence and incidence of laying hen fatty liver hemorrhage syndrome through nutritional intervention means. Therefore, it is urgent to construct a stable, reliable and efficient laying hen fatty liver hemorrhage syndrome disease model to systematically clarify its pathological mechanism and provide a key experimental basis for formulating targeted prevention strategies and developing effective treatment means.

[0003] Currently, in order to simulate the fatty liver disease characteristics of different etiologies and pathological stages, the research on the construction of animal fatty liver models has included multiple aspects such as diet induction, gene editing, drug intervention, combined induction and in vitro cell models. Nevertheless, in the research on the disease modeling of laying hen fatty liver hemorrhage syndrome, the construction methods still remain in relatively traditional methods such as diet induction and hormone intervention. Although these methods can simulate some pathological phenomena such as partial energy metabolism disorders or abnormal lipid synthesis, it is difficult to reproduce the core pathological characteristics of laying hen fatty liver hemorrhage syndrome - especially the hepatic parenchymal hemorrhagic lesions caused by increased vascular fragility. In recent years, the disease model construction strategy based on protein overexpression technology has gradually highlighted its unique advantages. By precisely controlling the expression level of specific functional proteins, it provides a new idea for analyzing the multi-dimensional pathogenic mechanism of laying hen fatty liver hemorrhage syndrome. In this context, angiopoietin-like protein 3 (hereinafter referred to as ANGPTL3) has become an important target for constructing a specific model of laying hen fatty liver hemorrhage syndrome due to its dual role in lipid metabolism and vascular homeostasis regulation.

[0004] ANGPTL3 is a glycoprotein mainly secreted by the liver, which regulates plasma triglyceride (TG) and high-density lipoprotein (HDL) metabolism by inhibiting the activities of lipoprotein lipase (LPL) and endothelial lipase (EL). Studies have shown that the abnormal overexpression of ANGPTL3 can lead to abnormal lipid metabolism and promote the ectopic deposition of liver lipids. This mechanism is highly consistent with the pathological characteristics of excessive accumulation of liver triglycerides in laying hens with fatty liver hemorrhage syndrome. More importantly, ANGPTL3 can also affect the function of the hepatic sinusoidal endothelial barrier by regulating the expression of vascular endothelial cell adhesion molecules (such as VCAM-1), suggesting that it may play a key role in the hemorrhagic lesions of laying hens with fatty liver hemorrhage syndrome.

[0005] Chinese Patent CN 116286981 A discloses the construction of a humanized ANGPTL3 overexpression lipid metabolism disorder model in mice and rabbits, whose applicable scope is mammals. The formation of mammalian fatty liver is related to the accumulation of intrahepatic triglycerides caused by insulin resistance, very low density lipoprotein (VLDL) secretion disorder, and impaired lipid oxidation. The mammalian fatty liver model cannot accurately simulate various molecular pathological cascade reactions and other processes in the hepatocytes of high-yield laying hens. Summary of the Invention

[0006] In order to solve the problem that the existing mammalian fatty liver model cannot accurately simulate various molecular pathological cascade reactions and other processes in the hepatocytes of high-yield laying hens, the present invention provides a method for constructing an ANGPTL3 overexpression laying hen fatty liver hemorrhage syndrome model and its application, which provides a basis and a new direction for the construction of a laying hen fatty liver hemorrhage syndrome model, and further promotes the research on the pathological process, disease treatment, and prevention of laying hen fatty liver hemorrhage syndrome.

[0007] To achieve the above object, the present invention provides a method for constructing an ANGPTL3 overexpression laying hen fatty liver hemorrhage syndrome model, including the following steps:

[0008] (1) Integrate the transposon element carrying the chicken ANGPTL3 coding gene and the transposase gene into the adenovirus vector respectively, and obtain recombinant adenovirus using a virus packaging system;

[0009] (2) Punch holes in the chicken embryo at 4 days of embryonic development (E4) using the equatorial plane fenestration method, with a pore diameter of 5 mm;

[0010] (3) Introduce the recombinant adenovirus into the E4 chicken embryo body by microinjection, and the injection dose of the recombinant adenovirus is 2.5 μl / egg, and the virus titer is 1×10^10^ pfu / ml;

[0011] (4) Incubate to laying hens to prepare a laying hen fatty liver hemorrhage syndrome model.

[0012] The transposable element is the piggyBac transposon, and the transposase gene is a functional transposase gene.

[0013] The preparation of the recombinant adenovirus includes:

[0014] a. Amplify the full gene sequence of chicken ANGPTL3 from the cDNA of laying hen liver by PCR, and introduce restriction enzyme sites at both ends;

[0015] b. Insert the full gene sequence of chicken ANGPTL3 and the EGFP coding sequence between the ITRs of the transposon, connect a strong promoter CMV upstream, and add a polyA signal downstream;

[0016] c. Clone the puromycin resistance gene (puro) into another independent expression cassette and drive it by the mCMV promoter;

[0017] d. Generate a plasmid by homologous recombination and linearize it, and obtain a high-purity recombinant adenovirus by virus packaging and amplification.

[0018] The preparation of the microinjection needle includes:

[0019] A. Use a quartz glass capillary to pull a microinjection needle with a laser needle puller;

[0020] B. Adjust the tip diameter to 60 μm and perform sterilization treatment.

[0021] The equatorial plane windowing method includes:

[0022] ① Mark the windowing site on the equatorial plane of the hatching egg to avoid the area with dense blood vessels, and drill a hole with an electric grinding head to obtain a round hole with a diameter of about 5 mm;

[0023] ② Fix the windowing site with a double-sided adhesive ring and seal the injection hole with sealing glue.

[0024] Application of the ANGPTL3 overexpression laying hen fatty liver hemorrhage syndrome model constructed by the method in the preparation of a laying hen fatty liver hemorrhage syndrome disease model.

[0025] The model is used for:

[0026] i. Analyze the pathogenesis of laying hen fatty liver hemorrhage syndrome;

[0027] ii. Discover early diagnostic markers;

[0028] iii. Screen and develop poultry drugs.

[0029] The present invention has the following beneficial effects:

[0030] The present invention uses laying hens as model animals and proposes for the first time a method of constructing an animal disease model using protein overexpression technology in poultry animals. Compared with other animals (rats, mice, and primates), it has the advantages of better innovation and low cost. Although there are a large number of examples of constructing animal models through genetic engineering technology, most of the research subjects are mice, mice, and primates, and the methods for constructing poultry models still remain at the level of dietary induction and hormone intervention. The core pathological characteristics of fatty liver hemorrhagic syndrome in laying hens include excessive lipid deposition in the liver, oxidative stress, microvascular damage, and hemorrhagic lesions. These processes involve the synergistic effects of lipid metabolism imbalance and vascular homeostasis disorders. Traditional diet or hormone-induced models can only partially simulate a single pathological link (such as lipid accumulation), while protein overexpression technology can simultaneously reproduce the dual phenotypes of metabolic disorders and vascular damage by targeted regulation of the expression levels of specific functional proteins (such as ANGPTL3, etc.). For example, overexpression of chicken ANGPTL3 can not only inhibit lipoprotein lipase activity leading to lipid clearance disorders, but also aggravate the destruction of the hepatic sinusoidal barrier by regulating the function of vascular endothelial cells, thereby completely simulating the dynamic development process of fatty liver hemorrhagic syndrome in laying hens from lipotoxicity to hemorrhagic lesions.

[0031] In addition, in view of the high economic cost of the virus packaging system, in order to reduce the modeling cost and improve the universality of the research plan, so as to promote the widespread application of this technology in large-scale breeding scenarios, the present invention selects chicken embryos as the research object, and achieves cost control by reducing the virus injection dose, thereby constructing an animal model of fatty liver hemorrhagic syndrome in laying hens with protein overexpression, which is used to carry out research on pathological mechanisms, early diagnosis methods and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 HE staining comparison of liver sections of laying hens' fatty liver hemorrhagic syndrome model induced by high-fat diet, laying hens' fatty liver hemorrhagic syndrome model with overexpression of ANGPTL3, and control group laying hens.

[0033] Figure 2 Oil red O staining comparison of liver sections of laying hens induced by high-fat diet and fatty liver hemorrhagic syndrome model, ANGPTL3 overexpressing laying hens model and control group laying hens.

[0034] Figure 3 It is the ratio of the area occupied by lipid droplets in liver sections of laying hens in the fatty liver and hemorrhagic syndrome model induced by high-fat diet, the fatty liver and hemorrhagic syndrome model of laying hens with overexpression of chicken ANGPTL3, and the control group. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described in detail below in conjunction with embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0036] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0037] Example 1

[0038] 1.1 Incubating chicken embryos

[0039] (1) Select fresh Sanhuang chicken breeding eggs within 7 days after production from the farm. When selecting, pay attention that the selected breeding eggs have intact eggshells without cracks, clean surfaces, and no obvious feces or dirt attached.

[0040] (2) Moisten the gauze with the diluted bromogeramine solution and wipe the surface of the fertilized eggs to kill the pathogenic microorganisms on their surfaces. The concentration of the dilution used is 50 mg / L.

[0041] (3) Place the disinfected fertilized eggs with the big end up on the tray, let them stand for 30 minutes to dry naturally, and then mark them with a marker pen.

[0042] (4) Transfer the processed fertilized eggs to an automatic incubator and incubate them in an environment of 37.8 °C and 65% humidity for 4 days. Automatically turn the eggs every two hours, and the turning angle is 45°, to prevent the chicken embryos from adhering to the eggshell membrane and at the same time promote the uniform development of the yolk sac blood vessels.

[0043] 1.2 Preparation of recombinant adenovirus particles for injection

[0044] (1) Amplify the full gene sequence of chicken ANGPTL3 from the cDNA of laying hen liver by PCR and introduce restriction enzyme cleavage sites at both ends.

[0045] (2) Insert the amplified full gene sequence of chicken ANGPTL3 and the EGFP coding sequence between the ITRs of the piggyBac transposon, connect a strong promoter CMV upstream, and add a polyA signal downstream.

[0046] (3) Clone the puromycin resistance gene (puro) into another independent expression cassette and drive it by the mCMV promoter.

[0047] (4) Ensure the successful construction of the chicken ANGPTL3 plasmid by double enzyme digestion.

[0048] (5) The constructed plasmid and the adenovirus backbone plasmid are subjected to homologous recombination to generate

[0049] pAd-PB-CMV-ANGPTL3-mCMV-puro plasmid, which is linearized, and high-purity recombinant adenovirus is obtained by virus packaging and amplification.

[0050] (6) High-purity recombinant adenovirus containing the transposase gene sequence is constructed by the above method. After mixing the two viruses, chicken ANGPTL3 recombinant adenovirus particles for injection are obtained.

[0051] 1.3 Preparation of microinjection needles (pulling, breaking, and sterilization treatment)

[0052] The material of the microinjection needles used in this invention is quartz glass capillary. The whole preparation process includes two steps: pulling and breaking the needles.

[0053] Pulling the needles:

[0054] (1) Set the pulling parameters of the horizontal laser needle puller according to the glass type and the use of the needle tip (such as intracellular injection, embryo manipulation). The microinjection needles used in this invention are made of quartz, and the injection object is the body of E4 chicken embryos. The parameters are set as follows: HEAT450, FIL4, VEL40, DEL130, PUL150.

[0055] (2) When pulling the needles, first place the glass needle in the fixed position, adjust the position of the right needle tip and then tighten the screw to fix it. Adjust the glass needle through the safety buckle so that it passes through the laser heating part. After adjusting the position of the left needle tip, tighten the screw to fix it.

[0056] (3) After fixing the glass needle, execute the program. The instrument will first heat to the glass softening point, apply a small pulling force to narrow the neck of the glass needle, and then increase the temperature and the pulling force to break the neck and form a sharp tip.

[0057] Breaking the needles:

[0058] (1) Use a microforge to adjust the diameter of the needle tip.

[0059] (2) After fixing the glass needle at 60 μm, observe the shape of the needle tip under an inverted microscope, and adjust the position of the glass needle in the field of view to make it close to the high-temperature melting sphere.

[0060] (3) Gently step on the foot pedal to raise the temperature of the high-temperature melting sphere to 90 °C. Release the foot pedal instantly when the lower end of the glass needle touches the melting sphere, and at the same time adjust the position of the glass needle upward. Break the needle through the temperature and pulling force of the melting sphere to obtain a microinjection needle with a diameter of 60 μm.

[0061] (4) Wrap the prepared microinjection needles with kraft paper and place them in an autoclave together with other instruments for 20 minutes of treatment.

[0062] 1.4 Equatorial plane windowing method for hole punching

[0063] (1) Preparation of double-sided adhesive ring and sealing glue: Select a closed-cell foam double-sided adhesive with a thickness of 3.0 mm (width ≥ 2 cm), fold it along the long axis direction and press and bond it to form a double-layer tape with a thickness of 6.0, and cut the tape transversely into equal-length segments of 3.0 cm.

[0064] (2) Standardized operation for hole punching: Fix the tape segment on a rubber backing plate, vertically apply pressure to strike a metal hole punch to obtain a circular hole with a diameter of 1.5 cm. When punching holes, ensure that the holes are centered and there is no obvious tearing at the edges.

[0065] (3) Preparation of sealing glue: Place a transparent sealing glue (width 5 cm) on a cutting template and use a surgical blade (No. 11)

[0066] Cut it into square patches of 6.0 mm along the grid lines and store them in a sterile petri dish for later use.

[0067] (4) Sterilization treatment of instruments: Place disposable No. 11 carbon steel surgical blades, No. 23 carbon steel blades, straight non-toothed forceps, and microinjection needles together in a high-pressure steam sterilizer for sterilization. The sterilization parameters are: 121 °C, 103.4 kPa, 20 min.

[0068] (5) PBS solution pretreatment: Dilute the penicillin-streptomycin solution at a ratio of 1:100 with PBS and dispense it into 50 mL

[0069] centrifuge tubes, and place them in a constant temperature water bath (37 °C) for preheating for 30 min for later use.

[0070] (6) Select the hatching eggs on the 4th day, with the air chamber facing up, and use a marker pen to mark the windowing site on the equatorial plane avoiding the area with dense blood vessels.

[0071] (7) Use an electric grinding head to drill a hole perpendicular to the marked surface of the hatching egg to obtain a circular hole with a diameter of about 5 mm and expose the eggshell membrane. Then, use a 75% alcohol cotton ball to wipe the windowing area in a circular motion for disinfection treatment.

[0072] (8) Use sterile forceps to hold the double-sided adhesive ring so that the central hole can completely expose the eggshell windowing site, and gently press the edge of the adhesive ring to expel air bubbles.

[0073] 1.5 Microinjection of recombinant adenovirus particles

[0074] (1) Use a disposable sterile plastic dropper to suck the preheated PBS solution and vertically drop it into the circular hole of the double-sided adhesive ring so that the liquid surface completely covers the eggshell membrane.

[0075] (2) Use an 11-carbon steel blade at a 30° angle to the eggshell surface and make a "cross" incision along the edge of the round hole. The incision length is about 2 mm, and the incision depth is limited to penetrating the eggshell membrane without damaging the chorioallantoic membrane.

[0076] (3) Use straight-toothed forceps without teeth to clamp the incision edge and peel the eggshell membrane along the tangent direction to expose the interior of the egg.

[0077] (4) Gently rotate the eggshell under the microscope to find and observe the state of the chicken embryo, and focus the microscope on the head to heart region of the chicken embryo. It can be observed that the chicken embryo heart beats rhythmically.

[0078] (5) Select a microinjection needle and insert it into the chicken embryo body at a 45° angle from the side of the embryo trunk, avoiding the heart and yolk sac blood vessels. Gently rotate the eggshell. If the embryo drifts slightly with the liquid and there is no obvious bleeding, it is confirmed that the tip of the needle is at the injection target site.

[0079] (6) Use a syringe to inject 2.5 μl of recombinant adenovirus into the chicken embryo body and maintain the pressure for 1 s. After injection, withdraw the needle vertically. Observe that there is no obvious abnormality in the embryo and no obvious bleeding at the injection site.

[0080] (7) When sealing the membrane, align the sterile transparent sealing film with the opening at the equatorial plane and evenly press it with a plastic rod under vertical pressure to ensure that the film fits seamlessly with the eggshell.

[0081] (8) Stack the second layer of sealing film, with the direction crossing the first layer at a 45° angle, and also evenly press it with vertical pressure. After pressing, there is no obvious warping at the edge of the film.

[0082] (9) Use a 23-carbon steel blade to vertically cut the double-sided adhesive ring horizontally, and at the same time use forceps to clamp the edge of the adhesive ring and slowly peel it off to avoid damaging the eggshell.

[0083] (10) Fix the sealed egg with sterile transparent tape in a "cross" shape to prevent the egg liquid from leaking out to the greatest extent. Finally, wipe the surface of the eggshell with a clean tissue to remove the residual liquid and other contaminants.

[0084] (11) Vertically fix the injected and sealed breeding eggs on the egg tray, with the air chamber facing up and the opening site at the equatorial plane facing left, at a 90° angle to the horizontal plane, and then transfer them to an automatic constant-temperature incubator. The temperature of the incubator is 37.8 °C, the humidity is 65%, and the eggs are automatically turned every two hours, with the turning angle being 45°.

[0085] 1.6 HE staining of liver tissue

[0086] (1) Take liver tissues from the experimental group (layer hens 90 days after microinjection and hatching), the positive control group (high-fat feeding), and the negative control (normal feeding). The tissue block size is 5×5×3 mm 3, placed in 4% paraformaldehyde for fixation for 24 h, and the volume ratio of the fixative to the tissue block was 20:1.

[0087] (2) Prepare tissue wax blocks by paraffin embedding, cut sections 5 μm thick, and bake them in a constant temperature oven at 60 °C for 2 h to ensure that the tissue is closely attached to the glass slide.

[0088] (3) Dewax with xylene: Immerse in xylene I for 15 min and then in xylene II for 15 min.

[0089] (4) Hydrate with gradient ethanol: 100% ethanol I for 5 min - 100% ethanol II for 5 min - 95% ethanol I for 5 min - 95% ethanol II

[0090] for 5 min - 80% ethanol for 5 min - 70% ethanol for 5 min - rinse with distilled water for 10 min.

[0091] (5) Immerse in hematoxylin staining solution for 5 - 8 min and then rinse with running water for 2 min to remove floating color.

[0092] (6) Differentiate with 1% hydrochloric acid ethanol for 2 s and then rinse with running water for 5 min, and then soak in tap water for 10 min to restore the blue color of the cell nucleus. (7) Immerse in eosin staining solution for 2 - 3 min and then rinse with running water for 2 min.

[0093] (8) Dehydrate with gradient ethanol: 70% ethanol for 20 s - 80% ethanol for 30 s - 90% ethanol for 1 s - 95% ethanol I for 1 min - 95% ethanol II for 1 min - 100% ethanol I for 1 min - 100% ethanol II for 1 min.

[0094] (9) Clear with xylene: Immerse in xylene I for 3 min and then in xylene II for 3 min.

[0095] (10) Finally, drop neutral resin on the surface of the tissue, slowly cover the cover glass, avoiding the generation of bubbles. Place the glass slide horizontally in a ventilated place away from light for curing for 24 h and then observe and photograph under an optical microscope.

[0096] (11) The results are as Figure 1 shown. Compared with the normal feeding group, both the high-fat feeding group and the ANGPTL3 overexpression group showed mild fat infiltration around the central vein area, and the fat droplets were more obvious in the high-fat feeding group and the ANGPTL3 overexpression group under high magnification.

[0097] 1.7 Oil Red O staining of liver tissue

[0098] (1) Take liver tissues from the experimental group (laying hens 90 days after microinjection and hatching), the positive control group (high-fat feeding), and the negative control (normal feeding). The size of the tissue block is 5×5×3 mm 3, placed in 4% paraformaldehyde for fixation for 24 h, and the volume ratio of the fixative to the tissue block was 20:1.

[0099] (2) Tissue paraffin blocks were prepared by paraffin embedding, and the paraffin blocks were sectioned into 5-μm thick sections with a cryostat microtome and attached to pre-cooled glass slides.

[0100] (3) After sectioning, the sections were soaked in distilled water at room temperature for 2 min to remove surface impurities, and finally immersed in 60% isopropanol pre-cooled at 4 °C for 2 min to dissolve residual lipids.

[0101] (4) The sections were immersed in Oil Red O staining solution and incubated at room temperature in the dark for 10 min.

[0102] (5) After quickly washing with 60% isopropanol (4 °C) for 5-10 s, the sections were rinsed 3 times with deionized water at 4 °C, 1 min each time.

[0103] (6) After counterstaining with hematoxylin staining solution for 5 min, the sections were rinsed with ice water and blued for 10 min.

[0104] (7) The excess water on the surface of the sections was gently wiped with filter paper, and pre-warmed glycerin jelly at 37 °C was added dropwise to cover the stained tissue, and then a coverslip was placed on it for sealing. Subsequently, observation and photographing were carried out with an optical microscope.

[0105] (8) The results were as Figure 2 shown. Compared with the normal feeding group, the Oil Red staining in the livers of the high-fat feeding group and the ANGPTL3 overexpression group was significantly obvious, indicating a large amount of lipid droplet deposition. Moreover, the Oil Red staining in the ANGPTL3 overexpression group was significantly deeper, indicating that the intensity of liver lipid deposition in the ANGPTL3 overexpression group was significantly higher than that in the high-fat feeding group, showing obvious fatty liver characteristics.

[0106] In summary, the present invention successfully constructed a chicken ANGPTL3 overexpression laying hen fatty liver and hemorrhagic syndrome (FLHS) model, and verified its effectiveness through histopathological analysis. Compared with the normal feeding group, obvious fat deposition and degeneration occurred in the livers of laying hens in the chicken ANGPTL3 overexpression group, indicating that overexpression of chicken ANGPTL3 exacerbated the pathological process of hepatic lipid ectopic deposition by interfering with lipid metabolism. The above results suggest that chicken ANGPTL3 may lead to the accumulation of triglyceride (TG) in hepatocytes by inhibiting the activity of lipoprotein lipase (LPL) or interfering with the secretion of very low density lipoprotein (VLDL), thus becoming a new therapeutic target for metabolic liver diseases.

[0107] Example 2

[0108] 2.1 Application in analyzing the pathogenesis of laying hen fatty liver and hemorrhagic syndrome

[0109] The recombinant adenovirus particles containing the transposon element carrying the chicken ANGPTL3-encoding gene and the transposase gene are used to overexpress ANGPTL3 in chicken embryos by microinjection. After the chicken embryos develop and hatch, a laying hen fatty liver hemorrhage syndrome model can be obtained. ANGPTL3 plays a key role in lipid metabolism and can inhibit the activity of lipoprotein lipase (LPL), thereby affecting lipid uptake and metabolism. By overexpressing ANGPTL3 in laying hens to construct a laying hen fatty liver hemorrhage syndrome model, the specific mechanism of its action in lipid metabolism can be studied more deeply.

[0110] 2.2 Application in the discovery of early diagnostic markers for laying hen fatty liver hemorrhage syndrome

[0111] This model can be used to identify biomarkers related to the pathogenesis of laying hen fatty liver hemorrhage syndrome. By comparing the gene expression, protein levels, and metabolites between the ANGPTL3 overexpression group and the control group, potential early diagnostic markers can be discovered. For example, the levels of specific lipid metabolites in plasma and the expression changes of specific genes in the liver can be detected to screen for potential early diagnostic markers.

[0112] 2.3 Application in the screening and development of poultry drugs

[0113] This model can also quickly evaluate the effects of drugs on the pathological characteristics of laying hen fatty liver hemorrhage syndrome, thereby screening out effective anti-disease drugs. At the same time, this model helps to study the effects of drugs on multiple targets, develop multi-target drugs, and improve the treatment effect. In addition, using this model can optimize drug doses and administration methods, evaluate the long-term effects of drugs, and provide references for clinical applications.

[0114] The above process details the preferred embodiments of the present invention. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for constructing a model of fatty liver hemorrhage syndrome in laying hens with overexpression of ANGPTL3, characterized in that, It includes the following steps: (1) Integrate the transposon element carrying the chicken ANGPTL3 encoding gene and the transposase gene into the adenovirus vector respectively, and use the virus packaging system to obtain recombinant adenovirus; (2) Punch holes in chicken embryos at 4 days of development (E4) by the equatorial plane windowing method, with a pore diameter of 2.5 mm; (3) Introduce the recombinant adenovirus into the body of E4 chicken embryos by microinjection. The injection dose of the recombinant adenovirus is 2.5 μl per chicken, and the virus titer is 1×10^10^ pfu / ml; (4) Incubate until laying hens are obtained to prepare a laying hen fatty liver hemorrhage syndrome model.

2. The construction method according to claim 1, characterized in that The transposon element is a piggyBac transposon, and the transposase gene is a functional transposase gene.

3. The construction method according to claim 1, characterized in that The preparation of the recombinant adenovirus includes: a. Amplify the full gene sequence of chicken ANGPTL3 from laying hen liver cDNA by PCR, and introduce restriction enzyme cutting sites at both ends; b. Insert the full gene sequence of chicken ANGPTL3 and the EGFP encoding sequence between the ITRs of the transposon, connect a strong promoter CMV upstream, and add a polyA signal downstream; c. Clone the puromycin resistance gene (puro) into another independent expression cassette and drive it by the mCMV promoter; d. Generate and linearize the plasmid by homologous recombination, and use virus packaging and amplification to obtain high-purity recombinant adenovirus.

4. The construction method according to claim 1, wherein The preparation of the microinjection needle includes: A. Use a quartz glass capillary tube to draw a microinjection needle with a laser puller; B. Adjust the tip diameter to 60 μm and perform sterilization treatment.

5. The construction method according to claim 1, characterized in that, The equatorial plane windowing method includes: ① Mark the windowing site on the equatorial plane of the breeding egg to avoid the area with dense blood vessels, and use an electric grinding head to drill a hole to obtain a round hole with a diameter of about 5 mm; ② Fix the windowing site with a double-sided adhesive ring and seal the injection hole with sealing glue.

6. Application of an ANGPTL3 overexpression laying hen fatty liver hemorrhage syndrome model constructed by the method according to any one of claims 1 to 5 in the preparation of a laying hen fatty liver hemorrhage syndrome disease model.

7. The application according to claim 6, wherein The model is used for: i. Analyze the pathogenesis of laying hen fatty liver hemorrhage syndrome; ii. Discover early diagnostic markers; iii. Screen and develop poultry drugs.

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