A method for constructing an angptl3 overexpression egg-laying chicken fatty liver hemorrhagic syndrome model and application thereof

Abstract

A method for constructing and applying an ANGPTL3 overexpression model of fatty liver hemorrhage syndrome in laying hens includes the following steps: (1) integrating the transposon element carrying the chicken ANGPTL3 encoding gene and the transposase gene into an adenovirus vector, and obtaining recombinant adenovirus using a viral packaging system; (2) punching holes in chicken embryos developed to day 4 (E4) using the equatorial fenestration method; (3) introducing the recombinant adenovirus into the body of the E4 chicken embryos via microinjection; (4) hatching into laying hens to prepare a model of fatty liver hemorrhage syndrome in laying hens. This invention is the first to construct an animal disease model in poultry through protein overexpression. The model of fatty liver hemorrhage syndrome in laying hens does not require high-fat feed, and has the characteristics of accurate animal model construction, significant fatty liver characterization, and high efficiency. It has significant application value in the research of fatty liver hemorrhage syndrome in laying hens and the development and screening of poultry drugs.

Description

Technical Field

[0001] This invention belongs to the field of model construction technology, specifically relating to a method for constructing and applying a chicken ANGPTL3 overexpression model of fatty liver hemorrhage syndrome. Background Technology

[0002] Hemorrhagic fatty liver syndrome (HFLS) in laying hens is a prevalent metabolic disease in the poultry industry today, similar to non-alcoholic fatty liver disease in humans. It is primarily caused by excessive lipid accumulation in the liver, leading to fatty degeneration of the hepatic liver. In the later stages, hens die from internal hemorrhage due to liver enlargement and rupture. Pathological features of HFLS in laying hens include hepatic fatty degeneration, hepatomegaly, and abdominal fat deposition. Because this disease is prevalent in high-producing laying hens during peak and late laying periods, and is characterized by its insidious and sudden onset, it can cause sudden death in high-producing hens, resulting in significant economic losses to the poultry industry. Furthermore, the sudden and insidious nature of HFLS makes it difficult to diagnose before onset, and currently, there are no other effective clinical diagnostic methods besides pathological autopsy, resulting in very limited preventative measures. Currently, there are no effective drugs or treatments for this disease; the only solution is to slow or reduce the incidence and morbidity of HFLS through nutritional intervention. Therefore, there is an urgent need to construct a stable, reliable, and efficient disease model of fatty liver hemorrhage syndrome in laying hens in order to systematically elucidate its pathological mechanism and provide a key experimental basis for formulating targeted prevention strategies and developing effective treatments.

[0003] Currently, research on animal fatty liver model construction to simulate the characteristics of fatty liver disease at different etiologies and stages includes various methods such as dietary induction, gene editing, drug intervention, mixed induction, and in vitro cell models. However, in disease modeling research on hemorrhagic fatty liver syndrome in laying hens, construction methods remain relatively traditional, such as dietary induction and hormone intervention. While these methods can simulate some pathological phenomena such as energy metabolism disorders or abnormal lipid synthesis, they struggle to reproduce the core pathological features of hemorrhagic fatty liver syndrome in laying hens—especially the hemorrhagic lesions in the liver parenchyma caused by increased vascular fragility. In recent years, disease model construction strategies based on protein overexpression technology have gradually demonstrated their unique advantages. By precisely manipulating the expression levels of specific functional proteins, they provide new insights into the multidimensional pathogenic mechanisms of hemorrhagic fatty liver syndrome in laying hens. Against this backdrop, angiopoietin-like protein 3 (ANGPTL3), due to its dual role in lipid metabolism and vascular homeostasis regulation, has become an important target for constructing specific models of hemorrhagic fatty liver syndrome in laying hens.

[0004] ANGPTL3 is a glycoprotein primarily secreted by the liver that regulates plasma triglyceride (TG) and high-density lipoprotein (HDL) metabolism by inhibiting the activity of lipoprotein lipase (LPL) and endothelial lipase (EL). Studies have shown that abnormally high expression of ANGPTL3 can lead to abnormal lipid metabolism and promote ectopic lipid deposition in the liver, a mechanism highly consistent with the pathological features of excessive triglyceride accumulation in hepatic fatty liver hemorrhage syndrome in laying hens. More importantly, ANGPTL3 can also affect the sinusoidal endothelial barrier function 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' fatty liver hemorrhage syndrome.

[0005] Chinese patent CN 116286981 A discloses the construction of a humanized ANGPTL3 overexpression lipid metabolism abnormality model in mice and rabbits. Its scope of application is mammals. Mammal fatty liver formation is related to insulin resistance, impaired secretion of very low density lipoprotein (VLDL), and triglyceride accumulation in the liver due to lipid oxidation. Mammal fatty liver models cannot accurately simulate various molecular pathological cascade reactions in the hepatocytes of high-laying hens. Summary of the Invention

[0006] To address the problem that existing mammalian fatty liver models cannot accurately simulate various molecular pathological cascade reactions in the hepatocytes of high-laying hens, this invention provides a method for constructing and applying an ANGPTL3-overexpressing model of fatty liver hemorrhage syndrome in laying hens. This provides a foundation and new direction for the construction of models of fatty liver hemorrhage syndrome in laying hens, thereby promoting research on the pathological process, treatment, and prevention of fatty liver hemorrhage syndrome in laying hens.

[0007] To achieve the above objectives, this invention provides a method for constructing an ANGPTL3-overexpressing hen fatty liver hemorrhage syndrome model, comprising the following steps:

[0008] (1) Transposon elements carrying the chicken ANGPTL3 encoding gene and transposase gene were integrated into adenovirus vectors, and recombinant adenovirus was obtained using a viral packaging system.

[0009] (2) For chicken embryos that have developed to day 4 (E4), holes are punched using the equatorial plane windowing method, with a hole diameter of 5 mm;

[0010] (3) The recombinant adenovirus was introduced into the body of an E4 chicken embryo by microinjection. The injection dose of the recombinant adenovirus was 2.5 μl / animal and the viral titer was 1×10^10^pfu / ml.

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

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

[0013] The preparation of the recombinant adenovirus includes:

[0014] a. The full gene sequence of chicken ANGPTL3 was amplified from cDNA in the liver of laying hens by PCR, and restriction enzyme sites were introduced at both ends;

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

[0016] c. The puromycin resistance gene (puro) was cloned into a separate expression cassette and driven by the mCMV promoter;

[0017] d. Generate plasmids through homologous recombination and linearize them, then use viral packaging and amplification to obtain high-purity recombinant adenovirus.

[0018] The preparation of the microinjection needle includes:

[0019] A. Using a quartz glass capillary tube, a laser needle drawing device is used to draw a micro-injection needle;

[0020] B. Adjust the needle tip diameter to 60μm and sterilize it.

[0021] The equatorial windowing method includes:

[0022] ① Mark the opening point on the equatorial surface of the hatching egg, avoiding the dense blood vessel area, and use an electric grinding head to drill a hole to obtain a round hole with a diameter of about 5mm;

[0023] ②Use double-sided adhesive rings to fix the window opening position, and seal the injection hole with sealing glue.

[0024] The ANGPTL3 overexpression model of hemorrhagic fatty liver syndrome in laying hens constructed by the method is applied to the preparation of disease models of hemorrhagic fatty liver syndrome in laying hens.

[0025] The model is used for:

[0026] i. To elucidate the pathogenesis of fatty liver hemorrhage syndrome in laying hens;

[0027] ii. Discovering early diagnostic biomarkers;

[0028] iii. Screening and developing poultry drugs.

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

[0030] This invention uses laying hens as a model animal and proposes for the first time a method for constructing animal disease models in poultry using protein overexpression technology. Compared with other animals (mice, mice, primates), this method has advantages in terms of innovation and low cost. While there are numerous examples of constructing animal models using genetic engineering, most studies focus on mice, mice, and primates; methods for constructing poultry models remain limited to dietary induction and hormone intervention. The core pathological features of fatty liver hemorrhage syndrome in laying hens include excessive lipid deposition in the liver, oxidative stress, microvascular damage, and hemorrhagic lesions. These processes involve the synergistic effect of lipid metabolism imbalance and vascular homeostasis dysregulation. Traditional dietary or hormone-induced models can only partially simulate a single pathological step (such as lipid accumulation), while protein overexpression technology, by targeting and regulating the expression levels of specific functional proteins (such as ANGPTL3), can simultaneously replicate the dual phenotypes of metabolic disorders and vascular damage. For example, overexpression of chicken ANGPTL3 can both inhibit lipoprotein lipase activity leading to lipid clearance impairment and exacerbate the destruction of the hepatic sinusoidal barrier by regulating vascular endothelial cell function, thus fully mimicking the dynamic development process of fatty liver hemorrhage syndrome in laying hens from lipotoxicity to hemorrhagic lesions.

[0031] In addition, given the high cost of virus packaging systems, in order to reduce modeling costs and improve the universality of research protocols, and to promote the widespread application of this technology in large-scale farming scenarios, this invention selects chicken embryos as the research object. By reducing the viral injection dosage, cost control is achieved, thereby constructing an animal model of protein overexpression in laying hens with fatty liver hemorrhage syndrome for research on pathological mechanisms, early diagnostic methods, and drug screening. Attached Figure Description

[0032] Figure 1 This study compares the liver sections (HE staining) of hemp liver tissue from a high-fat diet-induced fatty liver hemorrhage syndrome model in laying hens, a chicken ANGPTL3 overexpressing fatty liver hemorrhage syndrome model, and a control group.

[0033] Figure 2 This study compares the oil red O staining of liver sections from laying hens induced by a high-fat diet, a chicken ANGPTL3-overexpressing laying hens model of fatty liver hemorrhage syndrome, and a control group.

[0034] Figure 3 The ratio of lipid droplet area in liver slices from high-fat diet-induced fatty liver hemorrhage syndrome model, chicken ANGPTL3 overexpressing fatty liver hemorrhage syndrome model, and control group laying hens was used to determine the ratio of lipid droplet area in liver slices. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Example 1

[0038] 1.1 Hatching chicken embryos

[0039] (1) Select fresh Sanhuang chicken hatching eggs within 7 days of laying at the farm. When selecting, pay attention to the fact that the selected hatching eggs have intact shells without cracks, clean surfaces, and no obvious feces or dirt attached.

[0040] (2) Soak gauze with diluted benzalkonium chloride solution and wipe the surface of the fertilized egg to kill the pathogenic microorganisms on its surface. The concentration of the diluted solution used is 50 mg / L.

[0041] (3) Place the sterilized fertilized eggs with the larger end facing up on a tray and let them stand for 30 minutes to air dry naturally. Then mark them with a marker.

[0042] (4) Transfer the treated fertilized eggs to an automatic incubator and incubate them for 4 days at 37.8℃ and 65% humidity. Turn the eggs automatically every two hours at a 45° angle to prevent the chicken embryo from sticking to the eggshell membrane and to promote the uniform development of the yolk sac blood vessels.

[0043] 1.2 Preparation of recombinant adenovirus particles for injection

[0044] (1) The full gene sequence of chicken ANGPTL3 was amplified from the cDNA of laying hen liver by PCR, and restriction enzyme sites were introduced at both ends.

[0045] (2) The amplified chicken ANGPTL3 full gene sequence and EGFP coding sequence were inserted between the ITRs of the piggyBac transposon, with the strong promoter CMV connected upstream and the polyA signal added downstream.

[0046] (3) The puromycin resistance gene (puro) was cloned into another independent expression cassette and driven by the mCMV promoter.

[0047] (4) Double enzyme digestion ensured the successful construction of chicken ANGPTL3 plasmid.

[0048] (5) Generate a virus by homologous recombination of the constructed plasmid and the adenovirus backbone plasmid.

[0049] The pAd-PB-CMV-ANGPTL3-mCMV-puro plasmid was linearized, and high-purity recombinant adenovirus was obtained by viral packaging and amplification.

[0050] (6) A high-purity recombinant adenovirus containing a transposase gene sequence was constructed using the above method. The two viruses were mixed to obtain chicken ANGPTL3 recombinant adenovirus particles for injection.

[0051] 1.3 Preparation of microinjection needles (needle pulling, needle breakage, sterilization treatment)

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

[0053] Pull the pin:

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

[0055] (2) When drawing, the glass needle should be placed in the fixed position first. After adjusting the position of the right needle, 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, tighten the screw to fix it.

[0056] (3) After the glass needle is fixed, the program is executed. The instrument will first heat the glass to the softening point and apply a small pulling force to narrow the neck of the glass needle. Then the temperature is increased and the pulling force is increased to break the neck and form a tip.

[0057] Broken needle:

[0058] (1) Adjust the needle tip diameter using a micro forging furnace.

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

[0060] (3) Gently press the foot pedal to heat the high-temperature dissolving ball to 90°C. Release the foot pedal the moment the lower end of the glass needle touches the dissolving ball, and at the same time adjust the position of the glass needle upward. The needle breaks through the temperature and tension of the dissolving ball, and a micro-injection needle with a diameter of 60μm is obtained.

[0061] (4) Wrap the prepared microinjection needle in kraft paper and place it in an autoclave along with other instruments for 20 minutes.

[0062] 1.4 Equatorial Plane Windowing Method for Opening Holes

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

[0064] (2) Standardized operation of punching: Fix the tape segment to the rubber pad, apply vertical pressure to the metal punch to obtain a circular hole with a diameter of 1.5cm. When punching, ensure that the hole is centered and that there is no obvious tear at the edge.

[0065] (3) Sealing adhesive preparation: Place the transparent sealing adhesive (5cm wide) on the cutting template and use a scalpel blade (No. 11).

[0066] Cut the patches into 6.0mm square pieces along the grid lines and store them in sterile petri dishes for later use.

[0067] (4) Instrument sterilization: Disposable No. 11 carbon steel scalpel blades, No. 23 carbon steel scalpel blades, straight toothless forceps and microinjection needles were placed together in a high-pressure steam sterilizer for sterilization. The sterilization parameters were: 121℃, 103.4kPa, 20min.

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

[0069] Centrifuge tubes are placed in a constant temperature water bath (37℃) for 30 minutes to preheat before use.

[0070] (6) Select eggs that have been incubated for 4 days, with the air cell facing upwards, and use a marker to mark the opening point on the equatorial plane, avoiding the dense blood vessel area.

[0071] (7) Use an electric grinder to drill a hole perpendicular to the marked area on the 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 windowed area in a circular motion for disinfection.

[0072] (8) Use sterile tweezers to hold the double-sided adhesive ring so that the center hole can fully expose the opening point of the eggshell, and gently press the edge of the adhesive ring to remove air bubbles.

[0073] 1.5 Microinjection of recombinant adenovirus particles

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

[0075] (2) Use a No. 11 carbon steel blade to make a cross-shaped cut along the edge of the round hole at a 30° angle to the eggshell surface. The cut length is about 2mm, and the cut depth is limited to penetrating the eggshell membrane but not damaging the chorioallantoic membrane.

[0076] (3) Use straight-tipped toothless tweezers to hold the edge of the cut and peel off the eggshell membrane along the tangential direction to expose the inside 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 area from the head to the heart of the chicken embryo. You can observe that the chicken embryo's heart is beating rhythmically.

[0078] (5) Using a microinjection needle, insert it into the chicken embryo's body from the side of the embryo's trunk at a 45° angle, 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, then the needle tip is confirmed to be at the injection target site.

[0079] (6) 2.5 μl of recombinant adenovirus was injected into the chicken embryo body with a syringe and pressure was maintained for 1 second. After the injection was completed, the needle was withdrawn vertically. No obvious abnormalities were observed in the embryo and no obvious bleeding was observed at the injection site.

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

[0081] (8) Overlay a second layer of sealing film, with the direction intersecting the first layer at 45°. Press it evenly with vertical pressure, and the edge of the film should not be obviously lifted after pressing.

[0082] (9) Use a No. 23 carbon steel blade to cut the double-sided rubber ring vertically on the horizontal plane, and at the same time use tweezers to hold the edge of the rubber ring and slowly peel it off to avoid damaging the eggshell.

[0083] (10) Secure the sealant with sterile transparent tape in a cross shape to prevent egg liquid leakage to the greatest extent possible. Finally, wipe the eggshell surface with a clean paper towel to remove any residual liquid and other contaminants.

[0084] (11) The injected and sealed hatching eggs are vertically fixed in the egg tray with the air cell facing upward and the opening point on the equatorial plane facing to the left, forming a 90° angle with the horizontal plane. Then, they are transferred to an automatic constant temperature incubator. The incubator temperature is 37.8°C and the humidity is 65%. The eggs are automatically turned every two hours at a turning angle of 45°.

[0085] 1.6 HE staining of liver tissue

[0086] (1) Liver tissue was taken from the experimental group (laying hens 90 days after microinjection), the positive control group (high-fat feeding), and the negative control group (normal feeding). The size of the tissue block was 5×5×3mm. 3The tissue was fixed in 4% paraformaldehyde for 24 hours, with a fixative-to-tissue volume ratio of 20:1.

[0087] (2) Tissue blocks were prepared by paraffin embedding, 5μm thick sections were cut, and baked in a constant temperature oven at 60℃ for 2h to ensure that the tissue adhered closely to the glass slide.

[0088] (3) Xylene dewaxing: Immerse in xylene I for 15 minutes and then immerse in xylene II for 15 minutes.

[0089] (4) Gradient ethanol hydration: 100% ethanol I for 5 min — 100% ethanol II for 5 min — 95% ethanol I for 5 min — 95% ethanol II

[0090] 5 min — 80% ethanol 5 min — 70% ethanol 5 min — Distilled water immersion 10 min.

[0091] (5) Soak in hematoxylin dye solution for 5-8 minutes, then rinse with running water for 2 minutes to remove the floating color.

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

[0093] (8) Gradient ethanol dehydration: 70% ethanol 20s - 80% ethanol 30s - 90% ethanol 1s - 95% ethanol I 1min - 95% ethanol II 1min - 100% ethanol I 1min - 100% ethanol II 1min.

[0094] (9) Xylene transparent: Soak in xylene I for 3 minutes, then soak in xylene II for 3 minutes.

[0095] (10) Finally, add neutral resin to the tissue surface and slowly cover it with a coverslip, avoiding the formation of air bubbles. Place the slide horizontally in a ventilated place and cure in the dark for 24 hours before observing and photographing it under an optical microscope.

[0096] (11) The results are as follows Figure 1 As shown, compared with the normal feeding group, both the high-fat feeding group and the ANGPTL3 overexpression group showed mild fatty infiltration around the central venous area, while 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) Liver tissue was taken from the experimental group (laying hens 90 days after microinjection), the positive control group (high-fat feeding), and the negative control group (normal feeding). The size of the tissue block was 5×5×3mm. 3The tissue was fixed in 4% paraformaldehyde for 24 hours, with a fixative-to-tissue volume ratio of 20:1.

[0099] (2) Tissue blocks were prepared by paraffin embedding and sliced ​​into 5μm thick sections using a cryostat and attached to pre-cooled glass slides.

[0100] (3) After slicing, soak in room temperature distilled water for 2 minutes to remove surface impurities, and finally immerse in 60% isopropanol that has been pre-cooled at 4°C for 2 minutes to dissolve residual lipids.

[0101] (4) Immerse the slices in Oil Red O staining solution and incubate at room temperature in the dark for 10 min.

[0102] (5) Quickly soak in 60% isopropanol (4℃) for 5-10 seconds, then rinse with 4℃ deionized water 3 times, 1 minute each time.

[0103] (6) Counterstain with hematoxylin staining solution for 5 minutes, then rinse with ice water for 10 minutes to reverse the blue stain.

[0104] (7) Gently wipe the excess moisture from the surface of the slide with filter paper, add glycerin gelatin that has been preheated to 37°C to cover the stained tissue, and cover with a coverslip. Then observe and photograph with an optical microscope.

[0105] (8) The results are as follows Figure 2 As shown, compared with the normal feeding group, the high-fat feeding group and the ANGPTL3 overexpression group showed obvious oil red staining in the liver, indicating a large amount of lipid droplet deposition. Moreover, the oil red staining in the ANGPTL3 overexpression group was significantly darker, indicating that the intensity of lipid deposition in the liver of the ANGPTL3 overexpression group was significantly higher than that in the high-fat feeding group, showing obvious fatty liver characteristics.

[0106] In summary, this invention successfully constructed a FLHS (Fat Liver Hemorrhage Syndrome) model in laying hens with ANGPTL3 overexpression, and its effectiveness was verified through histopathological analysis. Compared with the normally fed group, the laying hens in the ANGPTL3 overexpression group showed significant fat deposition and degeneration in their livers, indicating that ANGPTL3 overexpression exacerbates the pathological process of ectopic lipid deposition in the liver by interfering with lipid metabolism. These results suggest that ANGPTL3 may become a novel therapeutic target for metabolic liver diseases by inhibiting lipoprotein lipase (LPL) activity or interfering with very low-density lipoprotein (VLDL) secretion, leading to the accumulation of triglycerides (TG) in hepatocytes.

[0107] Example 2

[0108] 2.1 Application in elucidating the pathogenesis of fatty liver hemorrhage syndrome in laying hens

[0109] Recombinant adenovirus particles containing transposon elements carrying the chicken ANGPTL3 encoding gene and transposase gene were microinjected to overexpress chicken ANGPTL3 in chicken embryos. After hatching, a model of hemorrhagic fatty liver syndrome in laying hens was obtained. ANGPTL3 plays a crucial role in lipid metabolism, inhibiting lipoprotein lipase (LPL) activity and thus affecting lipid uptake and metabolism. By establishing a model of hemorrhagic fatty liver syndrome in laying hens through ANGPTL3 overexpression, its specific mechanism of action in lipid metabolism can be studied more deeply.

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

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

[0112] 2.3 Application in screening and developing poultry drugs

[0113] This model can also rapidly assess the impact of drugs on the pathological features of fatty liver hemorrhage syndrome in laying hens, thereby screening for effective anti-disease drugs. Simultaneously, this model helps study the effects of drugs on multiple targets, develop multi-target drugs, and improve treatment efficacy. Furthermore, it can be used to optimize drug dosage and administration methods, evaluate long-term drug effects, and provide a reference for clinical applications.

[0114] The above process describes in detail the preferred embodiments of the present invention. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution 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 ANGPTL3 overexpression model of fatty liver hemorrhage syndrome in laying hens, characterized in that, Includes the following steps: (1) Transposon elements carrying the chicken ANGPTL3 encoding gene and transposase gene were integrated into adenovirus vectors, and recombinant adenovirus was obtained using a viral packaging system. (2) For chicken embryos that have developed to day 4 (E4), holes are punched using the equatorial windowing method, with a hole diameter of 5 mm; (3) The recombinant adenovirus was introduced into the body of an E4 chicken embryo via microinjection. The injection dose of the recombinant adenovirus was 2.5 μl / egg, and the viral titer was 1 × 10⁻⁶. 10 pfu / ml; (4) Hatching to laying hens 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 the 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. The full gene sequence of chicken ANGPTL3 was amplified from cDNA in the liver of laying hens by PCR, and restriction enzyme sites were introduced at both ends; b. Insert the chicken ANGPTL3 full gene sequence and EGFP coding sequence between the ITRs of the transposon, connect the strong promoter CMV upstream, and add the polyA signal downstream; c. The puromycin resistance gene was cloned into a separate expression cassette and driven by the mCMV promoter; d. Generate plasmids through homologous recombination and linearize them, then use viral packaging and amplification to obtain high-purity recombinant adenovirus.

4. The construction method according to claim 1, characterized in that, The preparation of microinjection needles includes: A. Using a quartz glass capillary tube, a laser needle drawing device is used to draw a micro-injection needle; B. Adjust the needle tip diameter to 60μm and sterilize it.

5. The construction method according to claim 1, characterized in that, The equatorial windowing method includes: ① Mark the opening point on the equatorial surface of the hatching egg, avoiding the dense blood vessel area, and use an electric grinding head to drill a hole to obtain a circular hole with a diameter of 5mm; ②Use double-sided adhesive rings to fix the window opening position, and seal the injection hole with sealing glue.

6. The application of the ANGPTL3 overexpression model of hemorrhagic fatty liver syndrome in laying hens constructed according to any one of claims 1 to 5 in the preparation of a disease model of hemorrhagic fatty liver syndrome in laying hens.

7. The application according to claim 6, characterized in that, The model is used for: i. To elucidate the pathogenesis of fatty liver hemorrhage syndrome in laying hens; ii. Discovering early diagnostic biomarkers; iii. Screening and developing poultry drugs.

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