Methods for manufacturing target proteins having active-related manufacturing obstacles using avian oviduct bioreactors, and vectors, nucleic acid constructs, and fusion proteins therefor

Abstract

This invention provides a method for manufacturing target proteins with activity-related manufacturing barriers using an avian oviduct bioreactor, as well as the method's vector, nucleic acid construct, and fusion protein. The method includes: constructing an expression unit encoding a fusion protein of the target protein and a removable production status control module; stably integrating this unit into the genome of avian germline cells or their precursor cells to obtain genetically modified poultry capable of germline transmission of the expression unit; expressing the fusion protein in the oviduct of female offspring and secreting it into egg white, wherein the production status control module reduces the target biological activity of the target protein during the manufacturing stage to reduce host damage and / or improve manufacturing tolerance; recovering the fusion protein from eggs or egg white, and removing the production status control module post-harvest by protease cleavage to restore or enhance the target biological activity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for manufacturing target proteins with activity-related manufacturing barriers using an avian oviduct bioreactor, as well as the carriers, nucleic acid constructs, and fusion proteins thereof. Background Technology

[0002] Thrombin-like enzymes (TLEs) are a class of serine proteases from snake venom that primarily act on fibrinogen. Different TLEs exhibit varying substrate selectivity and pharmacological effects; some TLEs can selectively cleave and continuously consume fibrinogen, producing fibrinolytic, anticoagulant, or antithrombotic effects. Anclobetaase, batroxobin, and other TLEs have been developed or investigated as fibrinolytic drugs, and other snake venom-derived proteases are being explored for use in thrombosis and vascular-related diseases. Despite the potential medicinal value of these proteins, their stable, large-scale, and quality-controlled preparation remains challenging.

[0003] Traditional preparation methods typically involve isolating and purifying TLE from natural snake venom, which carries risks such as limited raw material supply, batch-to-batch variations, and co-purification with other toxins. Recombinant expression holds promise for improving production scale and consistency, but different expression systems have their limitations: prokaryotic systems generally lack eukaryotic secretion pathways and natural glycosylation capabilities, and complex disulfide-bonded proteins are prone to misfolding or inclusion body formation; yeast systems may produce glycoforms different from natural proteins and may experience protein degradation or expression fluctuations; while mammalian cell systems possess relatively complete secretion and post-translational processing capabilities, production costs and scale-up requirements are high. For a specific target protein, one or more factors among glycosylation, disulfide bond formation, folding, and secretion may limit yield or activity.

[0004] Besides folding and post-translational processing, the biological activity of the target protein itself can also pose a manufacturing obstacle. For example, if a protease prematurely exhibits activity within the host cell or during secretion, it may damage the cell, interfere with secretion pathways, or induce a stress response. Therefore, even if the expression system can support the folding of the target protein, it is still difficult to achieve stable, high-level production if the target biological activity impairs the producing cell or the host organism. Consequently, it is necessary to reduce or isolate the target biological activity during the manufacturing stage and restore it after product harvesting.

[0005] Avian oviducts possess a strong capacity for secretory protein synthesis, and egg white is easily collected repeatedly and non-invasively. Stable integration of exogenous expression units into the avian genome, especially site-specific integration into active oviduct loci such as ovalbumin (OVA), allows for the expression and accumulation of exogenous proteins in the oviduct and egg white. Gene editing technologies such as CRISPR / Cas provide the means to construct avian bioreactors with well-defined genetic structures. However, simply increasing expression and secretion levels is insufficient to fully address the host damage and decreased tolerance caused by the activity of the target protein itself. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for manufacturing a target protein with activity-related manufacturing barriers using an avian oviduct bioreactor. The method comprises: (1) providing an expression unit encoding a fusion protein, the fusion protein comprising a target protein and a removable production state control module linked to the target protein; (2) stably integrating the expression unit into the genome of an avian reproductive cell or its precursor cell, and obtaining genetically modified poultry capable of germline transmission of the expression unit from the cells; and (3) expressing the fusion protein in oviduct tissue in female offspring carrying the expression unit and dispersing it. The fusion protein is secreted into the egg white of a poultry egg, wherein, under the same manufacturing conditions, compared with the corresponding target protein without the production state control module, the production state control module reduces the target bioactivity of the target protein during the biosynthesis, processing, secretion or accumulation of the fusion protein, thereby reducing host damage caused by the target bioactivity and / or increasing host manufacturing tolerance; and (4) the fusion protein is recovered from the poultry egg or egg white and then cut at a preset location to remove the production state control module, thereby obtaining the target protein, wherein the target bioactivity of the obtained target protein is restored or increased relative to before removal.

[0007] In one embodiment, the target bioactivity is selected from at least one of protein hydrolysis activity, nucleic acid degradation activity, membrane damage activity, pore formation activity, cell lysis activity, cell death induction activity, and abnormal signal activation activity.

[0008] In one embodiment, the poultry is a chicken, duck, goose, quail, pigeon, or turkey, preferably a chicken; and / or, the poultry reproductive cells or their precursor cells are primordial germ cells, primordial germ cell-derived cells, reproductive stem cells, or cells capable of forming reproductive chimeras.

[0009] In one embodiment, the expression unit is site-specifically integrated into an endogenous locus or its regulatory region in avian oviduct tissue that has transcriptional activity and / or enables the secretion of exogenous proteins into egg white.

[0010] In one embodiment, the endogenous locus is selected from any one of the ovalbumin locus, ovotransferrin locus, lysozyme locus, and ovomucin-related locus, preferably the chicken ovalbumin locus.

[0011] In one embodiment, the target protein is selected from any one of proteases, nucleases, cytotoxic proteins or peptides, membrane-disrupting proteins or peptides, pore-forming proteins or peptides, cell-penetrating peptides, and myotoxin-like peptides.

[0012] In one embodiment, the target protein is a serine protease derived from snake venom, preferably a snake venom thrombin, more preferably an Agkistrodon halys enzyme or a functional fragment or variant that maintains its fibrinogen-associated enzyme activity.

[0013] In one embodiment, the production state control module is any one of small ubiquitin-like modified protein, ubiquitin, maltose-binding protein, glutathione S-transferase, thioredoxin, transcription anti-termination factor NusA Fc, albumin and its engineered variants, preferably a small ubiquitin-like modified protein.

[0014] In one embodiment, the amino acid sequence of the fusion protein has at least 95% homology with SEQ ID NO.1; preferably SEQ ID NO.1.

[0015] In one embodiment, the nucleotide sequence encoding the fusion protein has at least 95% homology with SEQ ID NO.2; preferably SEQ ID NO.2.

[0016] In one embodiment, the present invention provides a target protein prepared by the above-described method.

[0017] In one embodiment, the present invention provides a fusion protein for manufacturing a target protein with activity-related manufacturing barriers, the fusion protein comprising a target protein and a removable manufacturing state control module linked to the target protein, wherein: (a) the target biological activity of the target protein is capable of causing host damage and / or reducing host manufacturing tolerance in a predetermined avian host; (b) the manufacturing state control module reduces the target biological activity during the biosynthesis, processing, secretion, or accumulation of the fusion protein compared to a corresponding target protein without the manufacturing state control module; and (c) the manufacturing state control module can be removed by cutting after the formation, secretion, or harvesting of the fusion protein to restore or enhance the target biological activity relative to its state before removal.

[0018] In one embodiment, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding the aforementioned fusion protein and an expression regulatory element operatively linked to the nucleic acid sequence, and the nucleic acid construct being stably integrated into the genome of avian reproductive cells or their precursor cells, enabling female birds produced by the cells to express the fusion protein in oviduct tissue and secrete it into egg white.

[0019] In one embodiment, the nucleic acid construct further comprises at least one of a secretory signaling element, a fallopian tube-biased expression element, a homologous arm for site-specific integration of the genome, a selection marker, and a removable selection marker.

[0020] In one embodiment, the present invention provides a vector comprising the above-described nucleic acid construct.

[0021] In one embodiment, the present invention provides a genetically modified avian germ cell, wherein the genome of the germ cell has the aforementioned nucleic acid construct stably integrated therein.

[0022] In one embodiment, the present invention provides an egg or egg white containing the aforementioned fusion protein.

[0023] In one embodiment, the present invention provides the use of the above-described fusion protein, the above-described nucleic acid construct, the above-described vector, or the above-described germ cell in the manufacture of a target protein with activity-related manufacturing barriers using an avian oviduct bioreactor.

[0024] In this specification, "target protein with activity-related manufacturing barriers" refers to a protein or polypeptide whose target biological activity, in a predetermined avian host or in cells used to evaluate host tolerance, can cause host damage and / or reduce host manufacturing tolerance, thereby limiting its stable expression, processing, secretion, accumulation, or continuous production. The target biological activity includes, but is not limited to, proteolysis, nucleic acid degradation, membrane damage, pore formation, cell lysis, cell death induction, or activation by abnormal signals.

[0025] “Host damage” includes decreased cell viability, reduced cell number, inhibited proliferation, tissue damage, impaired development, impaired reproduction, or other detectable adverse changes; “manufacturing tolerance” refers to the host’s ability to maintain cell viability, growth, development, reproduction, or sustained production during the expression, processing, secretion, or accumulation of the target protein. Reduced host damage or increased manufacturing tolerance can be determined by comparing a fusion protein containing a production state control module with a corresponding target protein lacking said module under comparable expression conditions.

[0026] A "production status control module" refers to a molecular module that is linked to the target protein, can reduce the target biological activity of the target protein at least in part of the manufacturing process, and can be removed by a predetermined treatment after the fusion protein is formed, secreted, or harvested. Preferably, the module is covalently linked to the target protein to form a fusion protein and is linked to the target protein through a preset protease cleavage site.

[0027] "Production state" refers to the state in which the target protein exhibits reduced target biological activity during the manufacturing process due to the action of the production state control module. Compared to the corresponding target protein without the production state control module, this state is accompanied by reduced host damage caused by the target biological activity and / or increased host manufacturing tolerance. The target protein may include proteases, nucleases, cytotoxic proteins or peptides, membrane-disrupting proteins or peptides, pore-forming proteins or peptides, cell-penetrating peptides, or myotoxin-like peptides.

[0028] "Restoration or enhancement of target biological activity" means that the target biological activity of the target protein increases relative to its state before removal after the removal of the production status control module; this statement does not require that its activity reach the same level as that of the protein from the natural source.

[0029] Compared to existing technologies, this invention sets the biosynthesis of the target protein and postharvest activity recovery as interconnected manufacturing stages: target bioactivity is reduced during expression, secretion, and egg white accumulation to minimize host damage and / or improve manufacturing tolerance; the production status control module is then removed after harvesting to restore or enhance target bioactivity. This combination differs from fusion tag schemes that only aim to improve solubility, promote purification, or generally enhance expression. Stable integration and oviduct-biased expression facilitate continuous production and reduce exposure to non-target tissues; site-specific integration also yields production lines with more defined genetic structures.

[0030] This application presents a reversible production state control strategy based on small ubiquitin-like modified proteins (SUMO). SUMO fusion can improve the solubility and folding of some poorly expressed proteins; SUMO-specific proteases can cleave SUMO at designed linker boundaries, and under appropriate design, the cleaved target protein will have the desired N-terminus. This application further utilizes the role of SUMO in reducing HCA activity during the manufacturing stage: during the individual development, protein secretion, and egg white accumulation of transgenic chickens, SUMO-HCA maintains low target protease activity; after egg harvesting, SUMO is removed by cleavage with SUMO-specific proteases to obtain HCA with increased activity.

[0031] This application targets the snake venom thrombin HCA as a proof-of-concept, and integrates the SUMO-HCA fusion expression unit into the ovalbumin locus. The resulting transgenic traits are germline transmissible, and the fusion protein accumulates in egg white. Based on anti-SUMO ELISA, the SUMO-HCA fusion protein in a single egg is estimated to be approximately 320 mg; this value is an indirect estimate. No significant survival, reproductive, or histopathological abnormalities were observed in the tested transgenic hens during the observation period. After cleavage with SUMO-specific protease, the recombinant HCA exhibits thrombin-like catalytic activity and fibrinogen degradation activity, and shows preliminary antithrombotic effects in animal models.

[0032] This application combines oviduct-biased expression, stable genome integration, and SUMO-mediated reversible activity control into a single poultry manufacturing process, achieving separation between the manufacturing stage and the postharvest activity recovery stage. This implementation provides a repeatable poultry biomanufacturing scheme for proteins with activity-related manufacturing barriers.

[0033] Bioactive proteins that intrinsically damage host cells are typically difficult to manufacture on a large scale. While transgenic animals can produce recombinant proteins in secretions such as milk and egg white, potent proteases and other highly active molecules may still damage the synthesizing cells or the host organism due to premature activation. This application describes a method for synthesizing snake venom-like thrombin in transgenic hens through oviduct-biased expression and SUMO-mediated reversible activity control, obtaining the cleaved and activated target protein after egg white harvesting.

[0034] A key obstacle in protease manufacturing is the activity-related cytotoxicity it generates during biosynthesis. Direct expression of active proteases can reduce cell viability and affect secretory function. Cell experiments in this application show that SUMO-HCA has less impact on cell viability and morphology compared to direct expression of SUMO-free HCA; HCA activity is increased after SUMO removal via protease cleavage. Therefore, reducing activity during manufacturing and restoring activity post-harvest constitutes the core technical relationship of this system.

[0035] Site-specific integration of exogenous expression units into endogenous OVA loci allows for higher-level, oviduct-biased expression by leveraging the regulatory environment of these loci within the fallopian tube. Compared to random integration, site-specific integration helps reduce positional effects and results in a more defined genomic structure for engineered strains. Within the tissue range tested in this application, SUMO-HCA was not detected in tissues outside the fallopian tubes; gross observation and histological examination revealed no significant tissue abnormalities, indicating that the host possesses manufacturing tolerance under the described conditions.

[0036] Based on indirect estimation using anti-SUMO ELISA, the SUMO-HCA fusion protein in a single egg is approximately 320 mg. Eggs can be repeatedly collected from the same laying hen, and the secreted protein accumulates directly in the egg white; continuous sampling results indicate that the system can continuously produce SUMO-HCA during the observation period of this application.

[0037] The recombinant HCA, after SUMO removal, exhibited optimal catalytic conditions, kinetic parameters, and related functional activities similar to those of the natural snake venom-derived *Agkistrodon halys* enzyme under the detection conditions described in this application. Mass spectrometry results indicate glycoform heterogeneity in the recombinant protein. Previous studies have predicted that *Agkistrodon halys* enzyme contains multiple N-glycosylation sites; the recombinant HCA in this application showed multiple molecular weight signals, and the mass spectra changed after PNGase F treatment, supporting the presence of N-glycosylation modification in the recombinant protein. These results do not indicate that its glycoform is completely identical to that of the natural protein.

[0038] A carrageenan-induced tail thrombosis model in mice demonstrated that cleaved recombinant HCA retained its biological activity in vivo and exhibited preliminary antithrombotic effects. A poultry oviduct bioreactor can secrete target proteins into easily collected egg white and allows for repeated harvesting from laying hens.

[0039] This application combines reversible activity control, stable genomic integration, and oviduct-biased secretion into a modular manufacturing process. For other proteins, only those whose production state control module can reduce their target biological activity and improve host tolerance during the manufacturing stage, and whose target biological activity can be restored or enhanced after harvesting by removing the module, are considered target proteins with activity-related manufacturing barriers as described in this invention.

[0040] In summary, this application established a transgenic avian oviduct bioreactor based on SUMO-HCA as a proof-of-concept, and completed host tolerance comparison, induction of cleavage-related phenotypes, site-specific integration of OVA sites, germline transfer of primordial germ cells, oviduct-biased expression, continuous accumulation of egg white, postharvest cleavage, and functional validation during the manufacturing phase. This system provides a controllable avian biomanufacturing pathway for proteins with activity-related manufacturing barriers. Attached Figure Description

[0041] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments are now briefly described. The following drawings only show some embodiments of this application, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram of the donor plasmid structure of the present invention; Figure 2This is a schematic diagram of the doxycycline (DOX)-induced SUMO-HCA activation system. The EF1α promoter continuously drives the constitutive expression of the SUMO-HCA fusion protein; the TRE3G element can regulate the cleavage and removal of the SUMO tag by the Ulp1 protease under doxycycline induction. Figure 3 These are typical cell morphology diagrams of DF-1 cells after treatment with gradient concentrations of doxycycline (0-4 μg / mL); Figure 4 These are the results of quantitative detection of cell viability after doxycycline administration; the data were obtained from two independent DF-1 cell lines and are presented as mean ± standard deviation. Figure 5 The assay involves Western blot verification of SUMO-HCA protein expression and doxycycline-dependent SUMO-tagged enzyme digestion; β-actin is used as an internal control protein. Figure 6 This is a map of the SUMO-HCA fusion protein expression vector regulated by the EF1α promoter and the tagless natural HCA expression vector; Figure 7 This is a diagram showing the cell morphology of DF-1 cells after transfection with plasmids pSJ21–1 and pSJ21–2. Figure 8 yes Figure 7 The results of quantitative cell counts.

[0043] Figure 9 The results are from Western blot analysis of histidine (His) tag protein extracted from transfected cells; the molecular weight of the SUMO-HCA fusion protein is 44.9 kDa, and the molecular weight of the mature free HCA protein is 28.2 kDa. Figure 10 This is a schematic diagram of the targeted knock-in strategy for inserting the SUMO-HCA gene into exon 2 of the ovalbumin (OVA) gene; the diagram is labeled with homologous arm sequences and PCR primer sites (P1~P4) in the conjugate region. Figure 11 This is the Sanger sequencing data, which confirms that this insertion was seamlessly and accurately integrated via the homology-directed repair (HDR) pathway. Figure 12 This is a Western blot image showing the expression levels of ovalbumin (OVA) and SUMO-HCA proteins in fallopian tube tissue; Figure 13 This is an image showing the immunohistochemical staining results of OVA and SUMO-HCA proteins in a section of fallopian tube tissue; scale bar: 50 micrometers; Figure 14 This is a reverse transcription PCR (RT-PCR) diagram of correctly spliced ​​HCA transcripts in cultured epithelial cells. Figure 15 This is an immunoblot analysis of SUMO-HCA protein in cell lysate and cell supernatant culture medium; Figure 16 This is a graph showing the results of detecting SUMO-HCA fusion protein in egg white using Western blotting technology. Figure 17 This is the result of SDS-PAGE analysis after SUMO protease digestion. The electrophoresis results show mature HCA protein (molecular weight approximately 27.4 kDa). Figure 18 This is a graph showing the endotoxin content in the protein of this application. Detailed Implementation

[0044] To enable those skilled in the art to better understand the technical solutions of this application, the present invention will be further described below with reference to the accompanying drawings and embodiments. The described embodiments are only some embodiments of this application, and not all embodiments. Based on the disclosure of this application, other implementation methods obtained by those skilled in the art without creative effort should also fall within the protection scope of this application.

[0045] I. Materials and Methods

[0046] 1. Plasmid construction

[0047] To achieve efficient gene editing, this application utilizes the CRISPOR online website (http: / / crispor.gi.ucsc.edu / ) to predict the guide RNA with optimal cleavage efficiency targeting exon 2 of the chicken OVA gene. A Cas9 / sgRNA expression vector was constructed using the pX459 vector (Addgene, #62988) as the backbone. The donor plasmid contains upstream and downstream homologous arms (1000 bp 5' homologous arm, 800 bp 3' homologous arm) derived from the chicken OVA genomic locus. These homologous arm fragments were obtained through PCR amplification and purification. The serine protease HCA gene and SUMO gene were artificially synthesized after codon optimization and then amplified by PCR to obtain a fusion fragment. All fragments were ligated using a Gibson assembly kit (General Biotech, CL08050) to construct the donor plasmid, named pSJ30. The specific structure is described in [link to specific structure]. Figure 1 The relevant sequences are as follows.

[0048] Guide RNA nucleotide sequence SEQ ID NO.3: gagtcaccatgggctccatcgg Signal peptide sequence SEQ ID NO.4: HHANENIFYCPIAIMSALAMVYLGAK Fusion tag SUMO amino acid sequence SEQ ID NO.5: SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG SUMO proteases have extremely high sequence specificity, recognizing only the three-dimensional conformation of SUMO tags and cleaving them at precise sites. After cleavage, no extra amino acids remain at the N-terminus of the target protein, ensuring the native structure and activity of the product.

[0049] 2. Culture, transfection, and drug screening of chicken primordial germ cells (PGC) Primordial germ cells (PGCs) were isolated from the gonads of White Leghorn chicken embryos and cultured, transfected, screened, and enriched in vitro. Embryos hatched at 5.5–6 days of age were collected, and after removing surface yolk granules, transferred to PBS solution. The peritoneal cavity was opened, gonadal tissue was isolated, and transferred to 1.5 mL centrifuge tubes. 100 μL of 0.25% trypsin solution was added, and the tissue was incubated at 37 °C for 15 min for digestion. Serum-containing medium was added to terminate trypsin activity, and the cells were centrifuged at 500 g for 5 min at room temperature, discarding the supernatant. The cell pellet was resuspended in 400 μL of PGC medium and seeded into 24-well plates for incubation for 3 h. After embryonic fibroblasts adhered, the supernatant containing PGCs was aspirated and seeded into feeder cells (inactivated rat hepatic stellate cells, ATCC, CRL-1442) for culture.

[0050] PGC culture medium formulation: 40% conditioned medium, 46% Knockout DMEM (Gibco, 10829018), 7.5% fetal bovine serum (FBS, Gibco, 10099), 2.5% chicken serum (CBS, Gibco, 16110082), 100× non-essential amino acids (Gibco, 11140), 100× sodium pyruvate (Gibco, 11360), 200× penicillin-streptomycin mixture (Gibco, 15140), 1‰ β-mercaptoethanol, 1‰ recombinant human FGF (R&D, 34-FSE-025), 1‰ recombinant human SCF (R&D, 455-MC-050). Cells were cultured at 37 ℃ in a 5% CO2 incubator.

[0051] To achieve site-specific insertion of the exogenous gene into the OVA site in the PGC, 2 μg of donor plasmid and 2 μg of Cas9 / sgRNA expression plasmid were mixed in 1 mL of culture medium and 9.0 μL of FuGENE-HD transfection reagent (Promega, E2311) and co-transfected 1×10⁶ cells. 6 PGCs were cultured for 4 hours without a feeder layer, then the medium was replaced with complete PGC medium, and the cells were transferred to feeder layer cells for further culture. 24 hours after transfection, the cells were transferred to new culture dishes and screened with medium containing 1 mg / mL puromycin (InvivoGen, ant-pr-1). 48 hours after drug screening, positive PGCs were transferred to fresh puromycin-free medium for expansion culture.

[0052] 3. Preparation of OVA-HCA knock-in chickens The construction method of site-specific knock-in chicken is based on the published literature (Kim, YM, et al., The transgenicchicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancertherapeutic potential with enhanced Fc effector functions. Biomaterials. 167,58–68 (2018); Mukae, T., et al., Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens. Genes(Basel). 12, (2020); Kim, et al., Efficient production of recombinant humanadiponectin in egg white using genome edited chickens. Front Nutr. 9, 1068558(2022).; Cheng, Y., et al., CRISPR / Cas9-Mediated Chicken TBK1 Gene Knockoutand Its Essential Role in STING-Mediated IFN-β Induction in Chicken Cells.Front Immunol. 9, 3010 (2018)). Positive PGCs enriched after drug screening were collected, and the cell density was adjusted to 3000 cells / μL using PGC medium. After incubating the eggs for 54 h, a circular hole with a diameter of about 1 cm was made at the end of the eggshell opposite the air cell. The embryo floated to the opening, and 1 μL of cell suspension was injected into the blood vessels of the chicken embryo under a stereomicroscope. After sealing the hole, the eggs were incubated at 37.8 ℃ until day 21 to obtain G0 generation chimeric chickens.

[0053] 4. Chimeric chicken breeding G0 generation roosters were raised to sexual maturity, and their semen was collected for genomic DNA extraction and PCR identification. Roosters carrying the correct HCA knock-in allele were selected as positive. These positive roosters were then mated with wild-type hens to obtain G1 generation offspring. G1 generation hens were raised to laying age, and eggs containing exogenous protein were collected.

[0054] 5. Diethylstilbestrol (DES) treatment and separation of fallopian tube epithelial cells The DES treatment and oviduct sampling procedures were performed according to previous reports (Seo, et al., Physiological Effects of Diethylstilbestrol Exposure on the Development of the Chicken Oviduct. Journal of Animal Science and Technology. 51, 485-492 (2009); Sanders, MM and GS McKnight, Positive and negative regulatory elements control the steroid-responsive ovalbumin promoter. Biochemistry. 27, 6550-6557 (1988)). 100 mg of diethylstilbestrol powder (Sigma) was weighed, placed in a mold, and pressed under 5 tons of pressure for 5 min to prepare DES sustained-release pills. 15 mg of DES pills were subcutaneously implanted into the abdomen of 1-week-old female chicks. Ten days after treatment, the chicks were euthanized using 60%-70% CO2 asphyxiation, and the oviduct tissue was isolated for subsequent analysis. Untreated wild-type female chicks of the same age were selected as the control group. Three chicks were set up in each group to analyze the oviduct development characteristics and the effect of DES treatment.

[0055] The method for isolating chicken oviductal epithelial cells (cOEC) was based on the following references (Jung, KM, E. Yoo, and JYHan, An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells. Poult Sci. 103, 103723 (2024); Jung, JG, et al., Characterization and application of oviductal epithelial cells in vitro in Gallus domesticus. Biol Reprod. 85, 798–807 (2011).). Oviducts of 17-day-old Leghorn chickens were harvested. After tissue trimming, the inner epithelial layer was scraped off with a thin blade. The tissue was then placed in a digestion solution containing 0.25% trypsin (Gibco, 15050057) and 1 mg / mL collagenase P (Roche, 11213857001) for 30 min. Cells were dissociated and filtered through a 40 μm nylon sieve, and enzymatic digestion was terminated by adding 10% fetal bovine serum. After washing with PBS, the cells were then subjected to a 1×10⁻⁶ ppm solution. 6 Individual cells / well were inoculated into 6-well plates pre-coated with 0.1% matrix adhesive (Corning, 354227).

[0056] Oviduct epithelial cell culture medium: DMEM-F12 (Sigma, D8437), supplemented with 10% fetal bovine serum, 2% chicken serum, 200× penicillin-streptomycin, non-essential amino acids, β-mercaptoethanol, and 10 ng / mL epidermal growth factor (EGF) (Sigma, E9644); 5 mg / mL insulin-transferrin-selenium (ITS, Gibco, 41400045) and 0.5 mg / mL hydrocortisone (Merck, C-106) were also added. Culture conditions: 37 ℃, 5% CO2, 60% relative humidity. Cells were passaged into 25 cm² culture flasks when confluence reached 80%. During passage, add 1 mL of 0.25% trypsin to the T25 culture flask and incubate at 37 ℃ for 1–3 min; add 5 mL of complete culture medium to stop digestion and gently pipette to disperse the cells; seed into a new T25 flask at a passage ratio of 1:2, add culture medium to 5 mL, and incubate at 37 ℃ with 5% CO2.

[0057] 6. Reverse transcription PCR and real-time quantitative RT-PCR Total RNA was extracted using an RNA extraction kit (Meiji Bio, R4011-03), and cDNA was obtained using a reverse transcription kit (Aibotek, RK20429). HCA gene-specific primers were used for amplification and identification. Gene expression was quantified using a CFX96 real-time quantitative PCR instrument (Bio-Rad), with three biological replicates per group; relative expression levels were calculated using the ΔΔCt method. Primer sequences used are shown in Table 1.

[0058] Table 1. List of RT-qPCR primers

[0059] 7. Immunohistofluorescence staining Wild-type and OVA-HCA knock-in chicken oviduct tissues were fixed overnight in 4% paraformaldehyde (Biosharp, BL908A); the tissues were dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Embedded tissue sections (5 μm thick) were dried; sections were dewaxed, xylene removed, rinsed with distilled water, and subjected to antigen retrieval with citrate buffer. After washing three times with PBS, the sections were blocked for 1 h at room temperature with blocking buffer (Beyotime, P0102); rabbit anti-OVA primary antibody diluted 1:1000 (Abcam, ab18688) was added and incubated overnight at 4°C. After washing three times with PBS, fluorescently labeled secondary antibody diluted 1:500 (Thermo, A21207) was added and incubated for 1 h at room temperature; after washing again with PBS, DAPI-containing anti-fluorescence quenching mounting medium (Beyotime, P0131) was added, and images were acquired using a confocal microscope (Nikon).

[0060] 8. Western blot (protein immunoblotting) Total protein was extracted using cell / tissue lysis buffer (Huaxing Biotech, HX1798), and denatured at 95 °C for 5 min using 5× protein loading buffer (Beyotime, P0286). Protein samples were separated by 12% SDS-PAGE and transferred to PVDF membranes (Merck, IPVH00010). Blocking was performed with 5% skim milk at room temperature for 3 h. Primary antibodies against OVA, SUMO, His, GAPDH, or β-actin were added according to the target assay, and incubation was carried out overnight at 4 °C. After washing three times with TBST, HRP-labeled secondary antibody was added, and incubation was carried out at room temperature for 1 h. After another wash, ECL chemiluminescent substrate (Thermo, 34580) was added, and protein bands were acquired using a chemiluminescence imaging system (Bio-Rad).

[0061] 9. Protein concentration detection Protein concentrations were determined using the BCA protein quantification kit (Beyotime, P0010). Protein standards were prepared in concentration gradients of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL. 20 μL of the sample to be tested was added to each well. 200 μL of BCA working solution was prepared according to a 50:1 ratio (solution A:solution B), and incubated at 37 ℃ for 20–30 min. The absorbance at 562 nm was measured using a microplate reader, and the protein concentration was calculated based on the standard curve.

[0062] 10. Enzyme-linked immunosorbent assay (ELISA) This application uses ELISA to determine SUMO fusion protein (Shanghai Keabo Biotechnology, CB13062-Hu, standard range 0.25-8 ng / mL), fibrinogen (FIB) (BYabscience, BY-EM220705, standard range 0.25-8 ng / mL), fibrin degradation products (FDP) (Spbio, SP14622, standard range 250-4000 ng / mL), and tissue plasminogen activator (tPA) (Spbio, SP26543, standard range 0.9375-15 μg / mL). Purified and concentrated protein samples were directly tested; blood samples were anticoagulated with sodium citrate at a volume ratio of 9:1, thoroughly mixed, centrifuged at 1000 g for 20 min, and the supernatant was collected for analysis; urine samples were collected in sterile centrifuge tubes, centrifuged at 1000 g for 20 min, and the supernatant was collected for analysis.

[0063] Prepare gradient standards according to the kit instructions, setting up blank wells (without adding samples or enzyme-labeled reagents, all other operations are the same), standard wells, and sample wells. Add 50 μL of standard to the standard wells; add 40 μL of sample diluent to the sample wells, followed by 10 μL of the sample to be tested (final dilution 5-fold). Seal the plate with sealing film and incubate at 37 ℃ for 30 min; wash the plate 5 times, blot dry, add 50 μL of enzyme-labeled reagent to each well, and incubate at 37 ℃ for 30 min. Wash the plate again, blot dry, add 50 μL each of chromogenic reagent A and B to each well, vortex to mix, and develop color at 37 ℃ in the dark for 10 min. Add 50 μL of stop solution to stop the reaction, read the OD value at 450 nm using a microplate reader, and calculate the sample concentration based on the standard curve.

[0064] 11. Observation of Egg Structure and Morphology The micromorphology of the shell membrane in wild-type and OVA-HCA knock-in eggs was observed using field emission scanning electron microscopy (Zeiss Sigma). Eggshell samples were fixed overnight at 4 °C using Carno fixative (Phygene, PH0978); after graded ethanol dehydration and drying, they were observed and photographed under vacuum conditions using field emission scanning electron microscopy.

[0065] 12. Protein Extraction and Detection To extract HCA, egg white was first centrifuged to remove insoluble substances, and the supernatant was retained. 30% saturated ammonium sulfate (Solepro, A8821) was added to the supernatant and stirred thoroughly. After standing overnight at 4 °C, the mixture was centrifuged at 3200 g for 30 min at 4 °C, and the precipitate was collected. The precipitate was resuspended in PBS (Solepro, P1020), and then 45% ammonium sulfate was added to a final concentration of 50%. After incubation for 10 min, the mixture was centrifuged at 12000 g for 10 min. The resulting precipitate was resuspended in a buffer solution (pH 8.0) of 20 mM Tris-HCl and 50 mM NaCl, equivalent to four volumes of the original egg white. A standard curve was established using BSA standards, and the total protein concentration was roughly determined by absorbance at 595 nm. The protein solution was filtered through a 0.22 μm filter. The concentration of the fusion protein was determined using an anti-SUMO ELISA to indirectly estimate the content of the target fusion protein.

[0066] 13. Protease digestion, purification, and identification The fusion protein and SUMO protease were mixed at a mass ratio of 100:1 and digested overnight at 4 °C. The digestion products were purified using a Magnify Q HP strong anion exchange pre-packed column (Yisheng, 20471ES08). The column was equilibrated with 5 column volumes of binding buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.2); the target protein was loaded onto the column using a syringe. A linear gradient elution was performed using elution buffer (20 mM Tris-HCl, 50–200 mM NaCl, pH 5.0). The eluted fraction was collected, ultrafiltered and centrifuged at 4 °C, and the buffer was replaced with 20 mM Tris-HCl and 50 mM NaCl (pH 8.0). After concentration, the sample was analyzed by SDS-PAGE, and protein concentration was determined using a BCA kit. The sample was filtered through a 0.22 μm filter before mass spectrometry analysis.

[0067] Raw mass spectrometry data were processed using Progenesis QI software (v2.4, Waters) for peak alignment, mass spectrometry feature extraction, and peak intensity normalization. Component identification was performed based on the precise mass of molecular and fragment ions. N-glycans were released from natural Agkistrodon halys enzyme standard and recombinant HCA using the Rapid N-Glycan kit (Hzymes, #HBP003100L). The released N-glycans were fluorescently labeled, and quantification and structural analysis were achieved using fluorescence detection (FLR) and mass spectrometry, respectively.

[0068] 14. In vitro coagulation activity verification Blood was collected from healthy volunteers and anticoagulated with 3.8% (w / v) sodium citrate. Platelet-poor plasma (PPP) was prepared by centrifugation at 2500 g for 15 min at 4 ℃. Different doses of purified HCA were added to 0.2 mL of plasma, and coagulation time was recorded at 37 ℃. Human thrombin (2000 NIH units / mg) was used as a standard reference. One unit of coagulation activity was defined as the amount of protease required to produce a coagulation effect equivalent to that of standard thrombin; specific activity units were expressed as NIH units / mg protein. The minimum coagulation dose (MCD), i.e., the lowest amount of enzyme required to induce plasma coagulation within 60 s, was determined. Each dose group was tested independently in triplicate.

[0069] 15. Detection of fibrinogen degradation activity Purified HCA (50 μg / mL) was co-incubated with bovine fibrinogen (10 mg / mL) and plasminogen (Glu-Plg, 1 μmol / L) in 50 mM Tris-HCl and 0.1 M NaCl buffer (pH 7.8) at 37 °C to determine fibrinogen degradation activity. 10 μL of the degradation reaction solution was mixed with 40 μL of protein loading buffer, and after denaturation, the sample was detected by SDS-PAGE. Urokinase (Sigma, U4010) was used as a positive control; an equal volume of PBS incubation system served as a blank control.

[0070] 16. Protein stability test HCA was dissolved in Tris-HCl buffer containing 50 mM NaCl, and its thermal stability was assessed using a temperature gradient of 10–60 °C. Biological activity was measured after pre-incubation at different temperatures. pH stability was tested by dispersing HCA in buffer systems with pH values ​​ranging from 4–10 and incubating at 37 °C, followed by measurement of coagulation activity. Inhibitor sensitivity was assessed by incubating the protein with different concentrations of PMSF or EDTA at 37 °C for 1 h, followed by measurement of residual coagulation activity. All experiments were performed in triplicate.

[0071] 17. Enzyme kinetics assay HCA kinetic parameters were determined using the chromogenic substrate Bz-Phe-Val-Arg-pNA (MCE, HY-118959). Seven concentration gradient substrates (0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mmol / L) were prepared. The purified enzyme solution and each concentration substrate solution were pre-incubated at 37 °C for 5 min. After pre-incubation, the reaction mixture (200 μL / well) was added to a 96-well plate. Immediately after mixing, the absorbance change (ΔA / Δt) at 405 nm was continuously monitored using a microplate reader for 5–10 min; the linear range rate was taken as the initial reaction rate v0. Three technical replicates were set for each substrate concentration, and a blank control group without enzyme substrate was also included to subtract background. The absorbance change rate was converted to the molar reaction rate using the molar extinction coefficient of p-nitroaniline at 405 nm (ε = 9.6 × 10³ L・mol⁻¹・cm⁻¹). The substrate concentration [S] was fitted with the corresponding initial rate v0 according to the Michaelis-Menten equation to obtain K. m V max According to the formula kcat = V max / [E] t Calculate the catalytic constant kcat, where [E] t This represents the total enzyme concentration.

[0072] 18. Endotoxin testing The content of purified HCA endotoxin was determined using a commercially available endotoxin detection kit (Beyotime, C0276S). A standard curve was established using endotoxin standards at concentrations of 0.010, 0.025, 0.050, and 0.100 EU / mL. The procedure was as follows: 10 μL of endotoxin-free water, standard, or test sample was added sequentially to a pyrogen-free centrifuge tube, followed by 10 μL of endotoxin detection reagent; the tube was vortexed thoroughly and incubated at 37 °C for 25 min in the dark. 10 μL of colorimetric reagent was added, and the tube was vortexed again and incubated at 37 °C for 6 min. 50 μL of buffer A, buffer B, and buffer C were added sequentially; the mixture was thoroughly mixed, and the tube was incubated at room temperature for 5 min. The absorbance was read at 545 nm using a microplate reader. Each experiment was performed in triplicate to ensure data reliability. The level of natural Agkistrodon halys enzyme endotoxin was simultaneously measured as a control.

[0073] 19. Half-life detection Healthy adult ICR mice (5-6 weeks old, weighing 35-40 g) were selected and fasted for 4-6 hours before the experiment, with free access to water. Each mouse was injected with HCA via the tail vein at a dose of 5 μg / g body weight. Blood samples (50-100 μL) were collected at 0, 2, 4, 8, 12, and 24 hours post-administration. Blood samples were anticoagulated with 3.8% sodium citrate at a volume ratio of 9:1 and centrifuged at 4 ℃, 1000×g for 10 min. Urine samples were collected simultaneously. The HCA signal in the samples at each time point was detected using a BCA protein concentration assay kit, and time-concentration curves were plotted.

[0074] 20. Construction of thrombosis model and verification of antithrombotic effect Carrageenan (Yisheng, 60755ES10) was dissolved in physiological saline to prepare a 2% solution. The carrageenan solution was injected via the tail vein at a dose of 200 mg / kg, with the volume calculated at 100 μL per 10 g body weight. The length of the discolored segment of the tail was measured 24 h after injection to evaluate tail thrombosis. Different concentrations of HCA protein were injected intraperitoneally into the HCA-treated group, while the control group received an equal volume of physiological saline. Blood samples of 50–100 μL were collected from the orbital cavity at 2, 4, 8, 12, and 24 h after administration. The blood was anticoagulated with 3.8% sodium citrate at a 9:1 ratio, and the plasma was separated by centrifugation at 1000 g for 10 min at 4 ℃. The levels of plasma fibrinogen, fibrin degradation products (FDP), and tissue plasminogen activator (tPA) were quantitatively detected using ELISA. Tissue was taken from the tail 3 cm from the tip, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to prepare a specimen.

[0075] 21. Rat coagulation function test Prothrombin time (PT) and activated partial thromboplastin time (APTT) are commonly used indicators for evaluating coagulation function. This application investigated the effects of HCA on coagulation parameters in rats under different drug doses and durations. Blood samples were collected on days 1, 3, 5, 7, 9, 11, and 14 after drug administration, and PT and APTT were measured. Differences between groups were statistically analyzed.

[0076] 22. Statistical Analysis Statistical analysis and plotting were performed using GraphPad Prism software, and gene sequencing maps were visualized using SnapGene software. Unless otherwise specified, the biological and technical replicate numbers for each experiment are as stated in the corresponding figure captions or experimental records. Normally distributed data are expressed as mean ± standard deviation (mean ± SD). Independent samples t-tests were used for comparisons between two independent samples; one-way ANOVA was used for comparisons of three or more groups, with appropriate multiple comparisons performed according to the experimental design. The statistical significance threshold was set at p < 0.05.

[0077] II. Experimental Results

[0078] 1. A SUMO-based in vitro controllable activation system can achieve conditional activation of Agkistrodon halys enzyme (HCA). To construct a host cell-tolerant thrombin-like HCA expression protocol, this application first verified whether the SUMO fusion tag could inhibit enzyme activity during protein synthesis and simultaneously achieve conditional activation. We constructed a two-component inducible expression system in chicken DF-1 fibroblasts: the SUMO-HCA fusion protein was constitutively and continuously expressed, while the SUMO-specific protease Ulp1 was induced and regulated by doxycycline (DOX). Figure 2 Without DOX, cell morphology and viability remained normal; after treatment with gradient concentrations of DOX (0.5–4 μg / mL), the number of viable cells decreased in a dose-dependent manner. Figure 3 and Figure 4 This phenomenon conforms to the following mechanism: DOX induces the expression of Ulp1, Ulp1 cleaves the SUMO tag, releases active HCA, and subsequently produces cytotoxicity. Western blot results confirmed that cells stably express the SUMO-HCA fusion protein, and the protein cleavage process is strictly dependent on DOX induction. Figure 5 To more intuitively verify the enzyme activity blocking effect of the SUMO tag, we transfected DF-1 cells with the intact SUMO-HCA vector and the tagless free HCA vector, respectively. Figure 6 As can be seen from the comparison, cells expressing the SUMO-HCA fusion protein showed intact morphology and significantly higher survival rates than cells directly expressing unlabeled HCA. Figure 7 and Figure 8 Western blotting also detected target protein bands corresponding to both vectors. Figure 9 ).

[0079] The above results indicate that SUMO fusion reduces HCA-related cytotoxicity compared to direct expression of HCA without SUMO; SUMO-HCA can serve as a controlled state in the manufacturing process and releases HCA with enhanced activity after SUMO is cleaved.

[0080] In this invention, the amino acid sequence of the fusion protein SUMO-HCA is SEQ ID NO.1: SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGVIGGVECDINEHRFLVALYELTSMTFLCGGTLINQEWVVTAAHCDRLQLYLYIGMHDKYVKFDDEQGREPIEKYF YNCSNNLTTRDKDIMLIRLDRPVDNSTHIAPLSLPSRPPSVGSVCRVMGWGAISPSRDVLPDVPHCVNINLVNNAECRRAYPRLPATSRTLCAGVMQGGIDSCNRDSGGPLICDGQFQGVVNWGGNPCAQPNMPALYTKVYDYNDWIRSITAGNTTAACPPLIKVPLVDPHAC .

[0081] The nucleotide sequence encoding the fusion protein SUMO-HCA is SEQ ID NO.2:

[0082] 2. CRISPR / Cas9-mediated precise integration of SUMO-HCA into the endogenous ovalbumin (OVA) locus. To achieve tissue-specific expression in vivo, this application involves site-specific knock-in of the SUMO-HCA fusion expression cassette into the chicken endogenous ovalbumin (OVA) gene locus; this locus can drive efficient expression of the target protein in the oviduct medullaris. This application employs a CRISPR / Cas9 site-specific knock-in protocol to insert SUMO-HCA into exon 2 of the OVA gene. Figure 10 The selected sgRNA achieved a cleavage efficiency of 43.8% in DF-1 cells, meeting the requirements for homology-directed repair (HDR) experiments.

[0083] After co-transfection, antibiotic screening, and single-clonal amplification of primordial germ cells (PGCs), positive HDR clones were obtained by boundary PCR screening and verified by Sanger sequencing (Figure 11), confirming that the target fragment was precisely integrated at the junction of homologous arms at both ends. Two independent HDR-positive PGC cell lines were injected into recipient embryos, ultimately obtaining surviving G0 generation chicks (Table 2). Individuals carrying the correctly site-integrated allele were identified by PCR screening.

[0084] Table 2. Germline transfer efficiency and gene-edited chick production efficiency of ovalbumin site knock-in HCA (OVA-HCA KI) donor primordial germ cells (PGCs). 0318-50-pSJ30 41 35 6 14.63 % 7.31-9-pSJ30 23 20 3 13.04 % .

[0085] Subsequently, DNA was extracted from the semen of adult G0 generation roosters for PCR testing to assess the transgenic line transfer ability, and a stable genetic OVA-HCA knock-in line was successfully constructed.

[0086] 3. Oviduct-specific expression of SUMO-HCA in G1 knock-in chickens G0 generation roosters were crossbred with wild-type hens to obtain 86 surviving G1 generation offspring, of which 15 individuals carried the SUMO-HCA allele (Table 3). Six female positive individuals were obtained through CHD1Z / CHD1W genotyping.

[0087] Table 3. Reproductive productivity of G1 generation OVA-HCA knock-in chimeric chickens after testcrossing G0 generation OVA-HCA knock-in chimeric chickens with wild-type chickens. .

[0088] To accelerate oviduct development and maturation, heterozygous positive hens were treated with diethylstilbestrol (DES). DES increased oviduct tissue weight without significantly affecting hen body weight. Western blotting results confirmed that ovalbumin expression was upregulated in knock-in hens, and SUMO-HCA fusion protein was specifically detected. Figure 12 Immunostaining results showed proliferation of tubular glandular tissue in the fallopian tubes, with SUMO-HCA signal localized within secretory epithelial cells. Figure 13 ).

[0089] Reverse transcription PCR (RT-PCR) detected correctly spliced ​​HCA transcripts knocked into chicken oviduct epithelial cells. Figure 14 SUMO-HCA could be detected in both cell lysate and cell culture medium by Western blotting. Figure 15 This demonstrates that fallopian tube cells can express and secrete this fusion protein.

[0090] Reverse transcription PCR (RT-PCR) detected correctly spliced ​​HCA transcripts knocked into chicken oviduct epithelial cells. Figure 14 SUMO-HCA could be detected in both cell lysate and cell culture medium by Western blotting. Figure 15 This demonstrates that fallopian tube cells can express and secrete this fusion protein.

[0091] Tissue distribution analysis in adult hens revealed that SUMO-HCA was detected only in the oviduct tissue, with no positive signals in other major organs. Within the oviduct, the target protein showed the highest expression levels in the enlarged portion and shell gland region. Gross tissue observation and histological section analysis showed no obvious lesions in the tested tissues.

[0092] The results in summary confirm that SUMO-HCA is accurately expressed, and its expression level is limited to the fallopian tubes; under the experimental conditions, no significant morphological abnormalities or histopathological damage were observed in the tested tissues.

[0093] 4. Recombinant HCA sustained expression and efficient enrichment in egg white Compared with the wild-type control group, the egg white turbidity of eggs from hens knocked into with OVA-HCA was visibly increased, indicating changes in the composition of egg white proteins. Quantitative analysis showed no significant differences in egg weight, egg white volume, yolk weight, or eggshell-related indicators among different genotypes.

[0094] The total protein concentration in egg white of OVA-HCA knock-in eggs decreased slightly (P<0.01), but no significant changes were observed in the appearance and quality indicators of the eggs. During continuous observation of six adult hens, SUMO-HCA was continuously secreted and accumulated in the egg white. Since there is no fully validated HCA-specific ELISA, this application used an anti-SUMO ELISA for indirect quantification of the fusion protein. The ELISA standard curve was y = 0.1903x + 0.089, R² = 0.9991. The sample dilution factor was 1:3, and the egg white volume was approximately 25–30 mL. The estimated SUMO-HCA content per egg was approximately 320 mg. This result is an estimate based on the anti-SUMO signal and is not an absolute mass confirmed by orthogonal methods.

[0095] 5. Recovery and biochemical characterization of HCA derived from egg white Western blotting using anti-SUMO antibodies confirmed the presence of the target fusion protein in egg white. Figure 16 Egg white extract was treated with SUMO-specific protease, and the tag was removed to obtain mature HCA. SDS-PAGE showed a band at approximately 27.4 kDa, consistent with the theoretical molecular weight of mature HCA. Figure 17 After preliminary purification by anion exchange chromatography, the purity of the obtained HCA sample was approximately 55.3%. MALDI-TOF and liquid chromatography-mass spectrometry (LC-MS) analyses were used to verify the relevant peptides and molecular weights; PNGase F treatment results supported the presence of N-glycosylation modification in the protein.

[0096] Recombinant HCA, after SUMO excision, exhibited high catalytic activity under alkaline conditions (approximately pH 9) and at 35-40 °C. Its activity was inhibited by PMSF but relatively insensitive to EDTA, consistent with the characteristics of a serine protease. Nonlinear fitting against the substrate Bz-Phe-Val-Arg-pNA yielded Km = 0.28 ± 0.03 mmol / L and Vmax = 0.052 μmol·L⁻¹·min⁻¹. Under the same detection conditions, the kinetic parameters of the recombinant protein were close to those of the naturally occurring snake venom-derived Agkistrodon halys enzyme; a dose-dependent coagulation effect was also detected in human plasma. Endotoxin testing was performed on the purified samples used for subsequent activity assays. Endotoxin detection of HCA was performed using an endotoxin assay kit, and the standard curve was y = 5.5229x + 0.0741, R² = 0.9982. Substituting the absorbance values ​​of HCA and Acutobin into the above standard curve equation, the endotoxin content in purified HCA was calculated to be 0.048 ± 0.02 EU / mg, and the endotoxin content in the standard Acutobin was 0.027 ± 0.01 EU / mg. Both are far below the general endotoxin limit standard for biological products (generally ≤ 5 EU / mg), meeting the safety and quality control requirements for biological products. Figure 18 ).

[0097] 6. Recombinant HCA showed preliminary antithrombotic effects in animal models. In vitro fibrinogen degradation experiments showed that recombinant HCA preferentially cleaved the fibrinogen Aα chain, consistent with the thrombin-like activity characteristics reported in the literature.

[0098] After intravenous administration to ICR mice, plasma HCA signal peaked at 4 h and then gradually decreased at the set sampling time points; target protein-related signals were also detected in urine samples.

[0099] In a carrageenan-induced tail thrombosis model in mice, compared with the saline control group, mice in the HCA-treated group showed decreased plasma fibrinogen levels and increased fibrin degradation products. Tissue-type plasminogen activator (tPA) showed a transient increase 2-8 h after administration. Continuous observation for 48 h showed that the length of the thrombus-related discoloration segment in the mouse tail decreased with increasing dosage, consistent with tissue section results.

[0100] In a 14-day rat study, prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged in a dose- and time-related manner after HCA administration, indicating that HCA can affect coagulation function; however, this result alone is not equivalent to a complete evaluation of systemic toxicity.

[0101] In summary, recombinant HCA derived from eggs and processed by cutting retains fibrinogen degradation activity and exhibits preliminary antithrombotic effects in the animal models described.

[0102] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0103] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A method for manufacturing target proteins with activity-related manufacturing barriers using an avian oviduct bioreactor, characterized in that, The method includes: (1) Provides an expression unit encoding a fusion protein, the fusion protein comprising a target protein and a removable production state control module connected to the target protein; (2) The expression unit is stably integrated into the genome of avian reproductive cells or their precursor cells, and the cells are used to obtain genetically modified poultry capable of germline transmission of the expression unit; (3) To express the fusion protein in oviduct tissue and secrete it into egg white of a female offspring carrying the expression unit, wherein, under the same manufacturing conditions, compared with the corresponding target protein without the manufacturing state control module, the manufacturing state control module reduces the target biological activity of the target protein during the biosynthesis, processing, secretion, or accumulation of the fusion protein, thereby reducing host damage caused by the target biological activity and / or increasing host manufacturing tolerance; and (4) The fusion protein is recovered from the poultry egg or egg white and then cut at a preset position to remove the production status control module, thereby obtaining the target protein. The target biological activity of the obtained target protein is restored or improved relative to the state before removal.

2. The method according to claim 1, characterized in that, The target bioactivity is selected from at least one of the following: proteolytic activity, nucleic acid degradation activity, membrane damage activity, pore formation activity, cell lysis activity, cell death induction activity, and abnormal signal activation activity.

3. The method according to claim 1 or 2, characterized in that, The poultry is chicken, duck, goose, quail, pigeon or turkey, preferably chicken; and / or, the poultry reproductive cells or their precursor cells are primordial germ cells, primordial germ cell-derived cells, reproductive stem cells or cells capable of forming reproductive chimeras.

4. The method according to any one of claims 1-3, characterized in that, The expression unit is site-specifically integrated into an endogenous locus or its regulatory region in avian oviduct tissue that has transcriptional activity and / or enables the secretion of exogenous proteins into egg white.

5. The method according to claim 4, characterized in that, The endogenous locus is selected from any one of the ovalbumin locus, ovotransferrin locus, lysozyme locus, and ovomucoid-related locus, preferably the chicken ovalbumin locus.

6. The method according to any one of claims 1-3, characterized in that, The target protein is selected from any one of the following: protease, nuclease, cytotoxic protein or polypeptide, membrane-disrupting protein or polypeptide, pore-forming protein or polypeptide, cell-penetrating peptide, and myotoxin-like polypeptide.

7. The method according to claim 6, characterized in that, The target protein is a serine protease derived from snake venom, preferably a snake venom thrombin, more preferably an Agkistrodon halys enzyme or a functional fragment or variant that maintains its fibrinogen-related enzyme activity.

8. The method according to any one of claims 1-7, characterized in that, The production status control module is any one of the following: small ubiquitin-like modified protein, ubiquitin, maltose-binding protein, glutathione S-transferase, thioredoxin, transcription anti-termination factor NusA Fc, albumin and its engineered variants, preferably a small ubiquitin-like modified protein.

9. The method according to claim 8, characterized in that, The amino acid sequence of the fusion protein has at least 95% homology with SEQ ID NO.1; preferably SEQ ID NO.

1.

10. The method according to claim 8, characterized in that, The nucleotide sequence encoding the fusion protein has at least 95% homology with SEQ ID NO.2; preferably SEQ ID NO.

2.

11. A target protein prepared by the method of any one of claims 1-10.

12. A fusion protein for manufacturing target proteins with activity-related manufacturing barriers, characterized in that, The fusion protein includes a target protein and a removable production status control module linked to the target protein, wherein: (a) The target biological activity of the target protein is capable of causing host damage and / or reducing host tolerance in a predetermined avian host; (b) Compared to a corresponding target protein without the production state control module, the production state control module reduces the target biological activity during the biosynthesis, processing, secretion, or accumulation of the fusion protein; and (c) The production status control module can be removed by cutting after the fusion protein is formed, secreted or harvested, so that the target bioactivity is restored or enhanced relative to before removal.

13. A nucleic acid construct, characterized in that, The nucleic acid construct comprises a nucleic acid sequence encoding the fusion protein of claim 12 and an expression regulatory element operatively linked to the nucleic acid sequence, and the nucleic acid construct is capable of stable integration into the genome of avian reproductive cells or their precursor cells, enabling female birds produced by the cells to express the fusion protein in oviduct tissue and secrete it into egg white.

14. The nucleic acid construct according to claim 13, characterized in that, The nucleic acid construct further includes at least one of a secretory signaling element, a fallopian tube-biased expression element, a homologous arm for site-specific genome integration, a selection marker, and a removable selection marker.

15. A carrier, characterized in that, The vector comprises the nucleic acid construct according to claim 13 or 14.

16. A genetically modified avian reproductive cell, characterized in that, The genome of the germ cell is stably integrated with the nucleic acid construct of claim 13 or 14.

17. A poultry egg or egg white, characterized in that, The egg or egg white contains the fusion protein as described in claim 12.

18. Use of the fusion protein of claim 12, the nucleic acid construct of claim 13 or 14, the vector of claim 15, or the germ cell of claim 16 in the manufacture of a target protein with activity-related manufacturing barriers using an avian oviduct bioreactor.

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