Preparation method and detection method of recombinant cat-omega interferon antiviral preparation

Abstract

The invention provides a preparation method and a detection method of a recombinant cat-omega interferon antiviral preparation, and the preparation method comprises the following steps: carrying out reverse transcription on total RNA (Ribonucleic Acid) of cat spleen to obtain cDNA (Complementary Deoxyribose Nucleic Acid); by taking the cDNA as a template, executing a PCR amplification program on the cat IFN-omega gene sequence by using the specific primer to obtain a PCR product; carrying out connection operation on the PCR product and a pGEM-T-Easy vector, and transferring the PCR product and the pGEM-T-Easy vector into DH5alpha competent cells; the method comprises the following steps: extracting positive clone plasmids which are verified to be correct from DH5alpha competent cells by using specific primers to obtain recombinant expression plasmids, carrying out double enzyme digestion on the recombinant expression plasmids and a pET30a vector by using NdeI and XhoI, and transferring obtained recovered target fragments into BL21 (DE3) competent cells to identify positive recombinant plasmids containing target genes; transferring the positive recombinant plasmid containing the target gene into BL21 (DE3) competent cells for bacterial colony culture, and performing induced expression on the obtained culture thalli; and after the induced expression is completed, preparing a preparation corresponding to the cat interferon from the cultured thalli. The preparation efficiency is at least improved.

Description

Technical Field

[0001] This invention relates to data processing technology, and more particularly to a method for preparing and detecting a recombinant feline-ω interferon antiviral agent. Background Technology

[0002] Feline viral diseases (such as feline panleukopenia, feline coronavirus disease, and feline herpesvirus infection) pose a serious threat to the health of cats, often leading to high morbidity and mortality rates.

[0003] Interferon, a cytokine with broad-spectrum antiviral and immunomodulatory functions, has shown significant effects in the prevention and treatment of feline viral diseases. Among them, feline ω-type interferon (IFN-ω) has become a research hotspot in the field of veterinary medicine due to its strong antiviral activity and few side effects. With the expansion of pet ownership and the increasing emphasis on pet health, the market demand for efficient, stable, and scalable feline interferon preparations is growing. How to achieve efficient preparation of feline interferon through genetic engineering technology has become a key research direction in the field of pet biomedicine. Currently, existing technologies are mostly limited to the crude extraction stage of recombinant interferon protein. Purification, stability enhancement, and formulation processes tailored to the characteristics of feline interferon have not been established. The extracted interferon protein is easily degraded and loses its activity rapidly, making it impossible to prepare stable formulations that meet medical application standards. This severely restricts the large-scale application of feline interferon in pet clinical treatment and reduces the preparation efficiency of feline interferon.

[0004] Therefore, there is an urgent need for a preparation method and detection method for recombinant feline-ω interferon antiviral agents that can improve the preparation efficiency of feline interferon. Summary of the Invention

[0005] In view of the above problems, the present invention is proposed to provide a method for preparing and detecting a recombinant feline-ω interferon antiviral agent that overcomes or at least partially solves the above problems.

[0006] According to one aspect of the present invention, a method for preparing a recombinant feline-ω interferon antiviral agent is provided, comprising the following steps: Total RNA extracted from the corresponding cat spleen was reverse transcribed to obtain cDNA; Specific primers were determined based on the feline IFN-ω gene sequence, and the cDNA was used as a template to perform PCR amplification on the feline IFN-ω gene sequence using the specific primers to obtain PCR products. The PCR product was ligated to the pGEM-T-Easy vector for a predetermined duration, and then transformed into DH5α competent cells after the ligation was completed. Using specific primers, positive clone plasmids were extracted from the DH5α competent cells to obtain recombinant expression plasmids. The recombinant expression plasmids and pET30a vector were double-digested with NdeI and XhoI. The recovered target fragments were then transformed into BL21(DE3) competent cells to identify positive recombinant plasmids containing the target gene. The positive recombinant plasmid containing the target gene was transformed into BL21(DE3) competent cells for colony culture, and the resulting cultured cells were induced to express the gene. After the induction expression was completed, the cultured bacterial cells were used to prepare a formulation corresponding to feline interferon.

[0007] Optionally, in the method according to the invention, determining specific primers based on the cat IFN-ω gene sequence includes: The obtained feline IFN-ω gene sequence was modified by removing the corresponding signal peptide and adding the start codon ATG at the 5' end. After adding the corresponding start codon ATG, restriction enzyme sites Nde and XhoI are introduced at the 5' end of the primers to obtain specific primers, wherein the specific primers include the upstream primer FeIFN-ωF and the downstream primer FeIFN-ωR.

[0008] Optionally, in the method according to the present invention, the PCR product is ligated with the pGEM-T-Easy vector for a continuously preset ligation time, and after the ligation operation is completed, it is transformed into DH5α competent cells, comprising: The PCR product was purified and recovered, and the purified and recovered PCR product was ligated with the pGEM-T-Easy vector at 16°C for 4 hours. After the ligation operation is completed, the ligated PCR product and pGEM-T-Easy vector are transformed into DH5α competent cells.

[0009] Optionally, in the method according to the present invention, a positive clone plasmid is extracted and verified using specific primers from the DH5α competent cells to obtain a recombinant expression plasmid, comprising: DH5α competent cells were seeded in liquid culture medium and cultured with shaking at 37°C and 200 r / min. Positive clone plasmids were extracted and verified from DH5α competent cells after oscillation culture using upstream primer FeIFN-ωF and downstream primer FeIFN-ωR.

[0010] Optionally, in the method according to the invention, the cultured bacterial cells are used to prepare a formulation corresponding to feline interferon, comprising: The cultured bacterial cells were disrupted, and the disrupted bacterial cells were washed with inclusion bodies using urea and Triton solution to obtain lysis products. The lysis products were separated by chromatography, and the proteins obtained after chromatography were replaced with refolding buffer using G-25 packing material. The refolding buffer solution was stirred within a temperature range of 2-8°C and a duration of 8-24 hours, and then filtered using Sephadex G-25 gel after the stirring was completed. The obtained filtered product was placed in PBS buffer, and the corresponding feline interferon was prepared based on the obtained FeIFN-ω protein stock solution.

[0011] Optionally, in the method according to the invention, the preparation of a formulation of the corresponding feline interferon based on the obtained FeIFN-ω protein stock solution includes: Phosphate, sucrose, and amino acids were formulated into a freeze-drying protectant. FeIFN-ω protein stock solution was added to the freeze-drying protectant and mixed evenly. The resulting feline interferon was then dispensed and freeze-dried.

[0012] According to another aspect of the present invention, a method for detecting feline interferon prepared based on the above-described method for preparing recombinant feline-ω interferon antiviral agents is provided, comprising the following steps: Once it is determined that the dry powder vial containing feline interferon is conveyed to the first detection area via a conveyor belt, the oscillation unit is controlled to oscillate the dry powder vial. The oscillation operation control unit corresponding to the first detection area performs image acquisition on the dry powder medicine bottle, and determines the first detection value of the corresponding agglomeration dimension based on the obtained first detection image. When it is determined that the dry powder medicine bottle is transferred from the first detection area to the second detection area, the tilting unit is controlled to tilt the dry powder medicine bottle. The response completes the tilt operation control, the second acquisition unit corresponding to the second detection area performs image acquisition on the dry powder medicine bottle, and determines the second detection value of the corresponding impurity dimension based on the obtained second detection image; The quality status of the corresponding dry powder medicine bottle is determined based on the first detection value and the second detection value.

[0013] Optionally, in the method according to the invention, determining that the vial containing feline interferon powder is conveyed to the first detection area via a conveyor belt includes: The conveying speed of the corresponding conveyor belt and the unit distance between the primary sensing unit and the secondary sensing unit set in the corresponding first detection area are determined, and the conveying time is determined based on the conveying speed and the unit distance. When it is determined that the primary gravity acquisition value output by the primary sensing unit at any acquisition moment is greater than the preset acquisition value, the secondary gravity acquisition value output by the secondary sensing unit corresponding to the transmission duration is obtained, starting from that acquisition moment. If both the primary gravity acquisition value and the secondary gravity acquisition value corresponding to the transmission duration are greater than the preset acquisition value, it is determined that the dry powder medicine bottle has been transmitted to the first detection area.

[0014] According to the present invention, the present invention effectively solves the core pain points of low efficiency in the preparation of feline interferon and difficulty in forming stable formulations in the prior art, and provides a reliable technical solution for the large-scale and standardized production of feline IFN-ω. In the gene cloning and recombinant plasmid construction stages, this invention significantly improves the efficiency of target gene acquisition and the success rate of recombinant vector construction through targeted optimization: specific primers designed based on the cat IFN-ω gene sequence can accurately bind to template cDNA, greatly reducing heterogeneous interference during PCR amplification and ensuring efficient acquisition of pure target gene PCR products; at the same time, by controlling the preset ligation time between the PCR product and the pGEM-T-Easy vector and selecting highly adaptable DH5α competent cells for transformation, the vector ligation efficiency and transformation success rate are effectively improved; then, the recombinant expression plasmid and pET30a vector are accurately spliced ​​by double digestion with NdeI and XhoI; finally, high-purity positive recombinant plasmids are obtained by identification with BL21(DE3) competent cells, laying a solid genetic foundation for subsequent efficient expression and avoiding the preparation stagnation problem caused by low gene amplification efficiency and recombinant construction failure in traditional technologies; In the recombinant expression and formulation conversion process, this invention achieves efficient integration from active protein to qualified formulation: BL21(DE3) competent cells are selected as the expression host, and the induction expression system under the strong promoter regulation can efficiently synthesize feline IFN-ω protein, significantly increasing the expression level of the target protein; more importantly, this invention breaks through the limitations of the existing technology of "emphasizing crude extraction and neglecting formulation", and directly connects to the formulation preparation process targeting the characteristics of feline interferon after induction expression. Through systematic purification, stability control and dosage form forming process, the problems of easy degradation and rapid loss of activity of interferon protein are effectively solved, ensuring that the final feline interferon formulation meets the standards for pet medical application, and realizing efficient transformation of the whole chain from gene to finished formulation; Furthermore, the preparation process involved in this invention is clear, highly operable, and the parameters of each step are controllable and highly repeatable, which greatly reduces human error. It not only significantly improves the preparation efficiency of single batches of feline interferon, but also meets the needs of large-scale industrial production, providing the market with sufficient and stable feline IFN-ω preparations, effectively improving the prevention and treatment of feline viral diseases, and has important pet medical application value and industrialization prospects. Attached Figure Description

[0015] Figure 1 A flowchart illustrating a method for preparing a recombinant feline-ω interferon antiviral agent according to an embodiment of the present invention is shown; Figure 2 A schematic diagram illustrating the PCR amplification of the cat IFN-ω gene in this embodiment is shown; Figure 3 This diagram illustrates the PCR identification of the positive recombinant plasmid pET30a-FeIFN-ω in this embodiment. Figure 4 This diagram illustrates the double enzyme digestion identification of the positive recombinant plasmid pET30a-FeIFN-ω in this embodiment. Figure 5 A schematic diagram showing the expression of the positive recombinant plasmid pET30a-FeIFN-ω in this embodiment is shown; Figure 6 This diagram illustrates the expression and purification of the positive recombinant plasmid pET30a-FeIFN-ω in this embodiment. Figure 7 This diagram illustrates the Western Blot identification process in this embodiment. Figure 8 A schematic diagram illustrating the statistical effects of different dosages in this embodiment is shown; Figure 9 A flowchart of a method for detecting feline interferon according to another embodiment of the present invention is shown; Figure 10 A schematic diagram of the dry powder medicine bottle in this embodiment is shown; Figure 11 A structural block diagram of a cat interferon quality detection system according to another embodiment of the present invention is shown. Detailed Implementation

[0016] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0017] To address the problems existing in the prior art, the inventors proposed the solution of this invention. One embodiment of this invention provides a method for preparing a recombinant feline-ω interferon antiviral agent, wherein... Figure 1 The flowchart for the preparation method of the corresponding recombinant feline-ω interferon antiviral agent is as follows: Figure 1As shown, the method includes steps S101-106, which specifically includes the following: S101. Reverse transcribe the total RNA extracted from the corresponding cat spleen to obtain cDNA; S102. Based on the cat IFN-ω gene sequence, specific primers are determined, and the cDNA is used as a template to perform a PCR amplification program on the cat IFN-ω gene sequence using the specific primers to obtain PCR products. S103. The PCR product is ligated with the pGEM-T-Easy vector for a continuously preset ligation time, and then transferred into DH5α competent cells after the ligation operation is completed. S104. Using specific primers, extract and verify the positive clone plasmids from the DH5α competent cells to obtain the recombinant expression plasmid. Double digest the recombinant expression plasmid and the pET30a vector with NdeI and XhoI, and transform the recovered target fragment into BL21(DE3) competent cells to identify the positive recombinant plasmid containing the target gene. S105. Transform the positive recombinant plasmid containing the target gene into BL21(DE3) competent cells for colony culture, and induce expression in the obtained cultured cells. S106. After the induction expression is completed, the cultured bacterial cells are used to prepare a formulation corresponding to feline interferon.

[0018] The following is a detailed description of the above steps in this embodiment: For S101, the acquisition of total RNA and cDNA can be achieved using the following techniques: First, collect fresh cat spleens, taking care to avoid damaging the spleen membrane. Under aseptic conditions, wash the spleen surface with PBS, remove the membrane, cut off spleen tissue blocks, grind them, add 50 ml of D-Hanks solution, and repeatedly pipette and grind the tissue. Then filter through a 150-mesh sieve 1-2 times, centrifuge the filtrate at 1000 r / min for 10 minutes, collect the cell pellet, add 2-3 ml of erythrocyte lysis buffer, treat on ice for 10-15 minutes to lyse the erythrocytes, centrifuge at 1000 r / min for 10 minutes, collect the cell pellet, wash twice with PBS, centrifuge at 1000 r / min for 5 minutes after each wash, discard the supernatant, and then adjust the cell concentration to 1×106 cells / ml with RPMI 1640 culture medium (containing penicillin and streptomycin) containing 10% fetal bovine serum. The inducing agent concanavalin A (ConA) was then added to the cell culture medium to a final concentration of 10 μg / ml. The cells were induced and cultured at 37℃ in a 5% CO2 incubator for 24 hours, and the lymphocyte status was observed. Cells in good condition were collected, gently pipetted to lift adherent cells, mixed thoroughly, and aliquoted into 10 ml centrifuge tubes. The tubes were centrifuged at 1000 r / min for 10 minutes, washed twice with PBS, and centrifuged at 1000 r / min for 5 minutes after each wash. The supernatant was discarded, and total RNA was extracted from the cells according to the RNA extraction kit instructions. Finally, using total RNA as a template and Oligo(dT) as a reverse transcription primer, cDNA was synthesized according to the reverse transcriptase instructions of Takara Bio Engineering (Dalian) Co., Ltd.

[0019] Regarding S102, firstly, the specific primers can be determined based on the following technical aspects: The obtained feline IFN-ω gene sequence was modified by removing the corresponding signal peptide and adding the start codon ATG at the 5' end. After adding the corresponding start codon ATG, restriction enzyme sites Nde and XhoI are introduced at the 5' end of the primers to obtain specific primers, wherein the specific primers include the upstream primer FeIFN-ωF and the downstream primer FeIFN-ωR.

[0020] For example, in this embodiment, based on the feline IFN-ω gene sequence (DQ420222.1, NM_001089304) registered in GenBank, the signal peptide was removed and the start codon ATG was added to the 5' end to design a pair of primers FeIFN-ωF and FeIFN-ωR, and restriction enzyme sites NdeI and XhoI were introduced at the 5' end of the primers, respectively. The primer sequences are as follows: upstream primer FeIFN-ωF: 5'-CGCCATATGTGTGCCCTGCC-'; downstream primer FeIFN-ωR: 5'-CCGCTCGAGTTAAGATGACGCC-3'.

[0021] Secondly, using the cDNA as a template and the specific primers to perform a PCR amplification procedure on the cat IFN-ω gene sequence to obtain the PCR product, this can be achieved based on the following technical content: Using cDNA as a template, the feline IFN-ω gene was amplified using specific primers. The PCR amplification program was as follows: 94℃ pre-denaturation for 4 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, 35 cycles; 72℃ extension for 10 minutes. The corresponding amplification products could be detected by 1% agarose gel electrophoresis, yielding bands of approximately 500 bp. Figure 2As shown, M is the DL2000 Marker; 1 is the feline IFN-ω gene; and 2 is the negative control.

[0022] For S103, the sequential operations based on the PCR product and the pGEM-T-Easy vector can be specifically described as follows: The PCR product was purified and recovered, and the purified and recovered PCR product was ligated with the pGEM-T-Easy vector at 16°C for 4 hours. After the ligation operation is completed, the ligated PCR product and pGEM-T-Easy vector are transformed into DH5α competent cells.

[0023] For example, in this embodiment, the PCR product can be purified and recovered according to the instructions of the gel recovery kit, and then the purified PCR product can be ligated with the pGEM-T-Easy vector at 16°C for 4 hours, and then transformed into DH5α competent cells.

[0024] For S104, firstly, the extraction and verification of correct positive clone plasmids from the DH5α competent cells using specific primers can be achieved based on the following steps: DH5α competent cells were seeded in liquid culture medium and cultured with shaking at 37°C and 200 r / min. Positive clone plasmids were extracted and verified from DH5α competent cells after oscillation culture using upstream primer FeIFN-ωF and downstream primer FeIFN-ωR.

[0025] For example, in this embodiment, several single colonies were picked and inoculated into 4 ml of LB liquid medium containing 50 μg / ml Amp, and cultured overnight at 37°C with shaking at 200 r / min. Plasmids were extracted according to the instructions of a commercial plasmid extraction kit. The extracted recombinant plasmids were amplified using primers FeIFN-ωF and FeIFN-ωR. The recombinant plasmids that were identified as positive were named pGEM-T-FeIFN-ω and sequenced.

[0026] Among these methods, sequence analysis software can be used to perform nucleotide sequence homology analysis between the obtained feline IFN-ω gene and the feline IFN-ω gene registered in GenBank and other animal IFN-ω genes.

[0027] Secondly, the recombinant expression plasmid and pET30a vector were double-digested with NdeI and XhoI, and the recovered target fragment was transformed into BL21(DE3) competent cells to identify positive recombinant plasmids containing the target gene. This can be achieved based on the following technical aspects: For example, the pGEM-T-FeIFN-ω plasmid and pET30a vector can be double-digested with NdeI and XhoI, respectively, and the approximately 500bp FeIFN-ω gene target fragment and the approximately 5200bp pET30a vector target fragment can be recovered. The ligation product can be transformed into DH5α competent cells. After screening and identification of positive results, the plasmid can be transformed into BL21(DE3) competent cells to identify positive recombinant plasmids containing the target gene. BL21(DE3) can be, for example, Escherichia coli.

[0028] For S105, the induction of expression can be carried out based on the following steps: For example, 0.5 μl of the positive recombinant plasmid pET30a-FeIFN-ω can be transduced into BL21(DE3) competent cells, with a control group containing an empty vector. The cells are evenly spread on LB agar plates containing 50 μg / ml Kan and incubated overnight at 37°C. Single colonies are selected for rejuvenation. When the cells are incubated at 37°C and 200 rpm until the OD600nm value reaches approximately 0.8, 1 mmol / L IPTG is added to induce expression. The expression status is verified by SDS-PAGE before and after induction.

[0029] It can be noted that the positive recombinant plasmid pET30a-FeIFN-ω can be used for PCR identification. Based on the identification results, it can be seen that the amplified gene size is consistent with the size of the cat IFN-ω gene, approximately 500 bp (e.g., Figure 3 As shown, M is the LDL2000 Marker; 1 is the recombinant plasmid PCR product; 2 is the negative control; 3 is the positive control); double enzyme digestion identification results (e.g. Figure 4 As shown in the figure, M is the LDL2000 Marker; 1 is the double digestion product; 2 is the plasmid DNA. The fragment size is consistent with the expected result, which means that the prokaryotic expression vector was successfully constructed. The expression of feline IFN-ω in recombinant Escherichia coli BL21(DE3) / pET30a-FeIFN-ω can be seen as follows: Figure 5 As shown, M represents protein maker; 1 represents uninduced cells; 2 represents cells induced for 3 hours; 3 represents cells induced for 4 hours; and 4 represents cells induced for 5 hours.

[0030] As can be seen, after SDS-PAGE electrophoresis and Coomassie brilliant blue staining, the results showed that the recombinant plasmid was efficiently expressed in BL21(DE3), with a molecular weight of approximately 19.3 kDa.

[0031] Furthermore, after induction of recombinant E. coli BL21(DE3) / pET30a-FeIFN-ω expression, the bacterial cells were prepared as inclusion bodies, lysed, and purified by gel filtration chromatography to obtain a recombinant protein with high purity. SDS-PAGE verification showed that the protein purity was >95% (e.g., Figure 6 As shown, M represents the protein marker; 1 is the bacterial cell lysis supernatant; 2 is the inclusion bodies after washing; 3 is the sample after chromatographic separation; and 4 is the purified protein. The protein concentration was 0.45 mg / ml. Western blot identification results showed that cat IFN-ω protein can specifically react with cat IFN-ω monoclonal antibody (e.g., ...). Figure 7 As shown, M is the protein marker; 1 is the purified protein.

[0032] Regarding step S106, the preparation of the corresponding feline interferon formulation based on the cultured bacterial cells obtained in S105 may further include the following steps: The cultured bacterial cells were disrupted, and the disrupted bacterial cells were washed with inclusion bodies using urea and Triton solution to obtain lysis products. The lysis products were separated by chromatography, and the proteins obtained after chromatography were replaced with refolding buffer using G-25 packing material. The refolding buffer solution was stirred within a temperature range of 2-8°C and a duration of 8-24 hours, and then filtered using Sephadex G-25 gel after the stirring was completed. The obtained filtered product was placed in PBS buffer, and the corresponding feline interferon was prepared based on the obtained FeIFN-ω protein stock solution.

[0033] For example, in this embodiment, after induction of expression, bacterial cells can be collected by centrifugation, resuspended in PBS, and homogenized using a high-pressure homogenizer; inclusion bodies are washed with urea and Triton solution; after lysis, the lysis products are separated by chromatography using Sephacryl S-200 medium; the chromatographically separated protein is replaced with refolding buffer using G-25 packing material, and after refolding at 2-8°C with slow stirring for 18-24 hours, the buffer is replaced with PBS using a Sephadex G-25 gel filtration chromatography column, and after aseptic treatment, the FeIFN-ω protein stock solution is obtained.

[0034] Based on the above, after obtaining the protein stock solution, the following further detection process can be performed: 1. Commercially available BCA kits can be used for detection, and the protein concentration in the sample can be calculated based on the linear regression equation; 2. Add 5×SDS loading buffer to the cat ω-interferon protein before and after purification, mix thoroughly, boil at 100℃ for 5 minutes, load 10 μl of the denatured sample, and identify by SDS-PAGE using 5% stacking gel and 15% separating gel. After electrophoresis, stain with Coomassie Brilliant Blue R-250 for 30 minutes, and then decolorize with methanol-glacial acetic acid destaining solution for 40 minutes (change the solution once in between). Observe the results. 3. After separating the purified protein by SDS-PAGE electrophoresis, take a suitable-sized polyvinylidene fluoride (PVDF) membrane, soak it in 100% methanol for 5 minutes, and then immerse it in the electroporation buffer. Take a suitable-sized filter paper and wet it with the electroporation buffer. Apply the buffer at a rate of 0.65 mA / cm² based on the gel area. 2 Apply current and electroporate for 1 hour; transfer the membrane to blocking buffer and block at room temperature for 1-2 hours; remove the membrane and wash with PBST for 5 minutes, repeat 3 times; place the membrane in an appropriate concentration of feline interferon polyclonal antibody and incubate at room temperature for 2 hours or at 37°C for 1 hour; wash with PBST for 5 minutes, repeat 3 times; place the membrane in an appropriate concentration of enzyme-labeled secondary antibody and incubate at room temperature for 1 hour or at 37°C for 0.5 hours; remove the membrane and wash with PBST for 5 minutes, repeat 3 times; absorb the water from the membrane surface, immerse in the chromogenic solution, and observe the results for Western blotting identification. 4. The cytopathic effect inhibition method can be used to determine the cat IFN-ω anti-VSV activity on CRFK.

[0035] Furthermore, in this embodiment, the above-mentioned "preparation of the corresponding feline interferon formulation based on the obtained FeIFN-ω protein stock solution" may also include the following steps: Phosphate, sucrose, and amino acids were formulated into a freeze-drying protectant. FeIFN-ω protein stock solution was added to the freeze-drying protectant and mixed evenly. The resulting feline interferon was then dispensed and freeze-dried.

[0036] For example, in this embodiment, reagents such as phosphate, sucrose, and amino acids are prepared into a lyophilization protectant according to a certain ratio. After filtration and sterilization, it is set aside. Take 50 ml of the protectant, add 50 million IU of recombinant feline ω-interferon stock solution, add sterile water to make up to 100 ml, and prepare a 5 million IU / ml semi-finished product. After uniform mixing, it is dispensed and lyophilized. The product is placed in an incubator at room temperature. After 1 hour, the product is lowered to -40°C and maintained for 5 hours; after 1 hour, the product is raised from -40°C to -20°C and maintained for 20 hours; after 0.5 hours, the product is raised from -20°C to 0°C and maintained for 1 hour; after 0.5 hours, the product is raised to 10°C and maintained for 5 hours; after 0.5 hours, the product is raised from 10°C to 25°C and maintained for 8 hours, and the lyophilization is completed.

[0037] In addition, further tests can be conducted on the properties and vacuum level, such as property, vacuum level, activity determination, residual moisture determination, and 37°C stability test.

[0038] It can be noted that the treatment trials of feline interferon obtained based on the preparation method of this embodiment can be specifically described as follows: Thirty healthy, susceptible cats aged 2-3 months were randomly divided into 6 groups of 5 each. Group 1 (n=5) served as the challenge control group, receiving intranasal inoculation with feline herpesvirus type I strain BJS01. Groups 2-6 served as the treatment groups, receiving the same intranasal inoculation with the virus at the same dose. After the onset of the disease, the cats were administered subcutaneous injections at doses of 250,000 IU / kg, 500,000 IU / kg, 1,000,000 IU / kg, 2,000,000 IU / kg, and 4,000,000 IU / kg, respectively, twice daily for 14 consecutive days. Clinical symptoms and recovery rates were statistically analyzed in each group.

[0039] It should also be noted that, for freeze-dried products, the test results are as shown in the following table of freeze-dried product test results. The product properties, residual moisture, vacuum degree, biological activity, and stability all meet the requirements.

[0040] Test Results Table for Freeze-Dried Products

[0041] Regarding the results of the FeIFN-ω lyophilized formulation treatment trial, healthy susceptible cats aged 2-3 months were inoculated intranasally with feline herpesvirus type I. After the onset of illness, different concentrations of the formulation were used for treatment. The corresponding experimental results are as follows: Figure 8 As shown, subcutaneous injection of doses higher than 1 million IU / kg can significantly improve the survival rate of cats infected with herpesvirus.

[0042] In summary, this embodiment effectively solves the core pain points of low efficiency in the preparation of feline interferon and difficulty in forming stable formulations in the prior art, and provides a reliable technical solution for the large-scale and standardized production of feline IFN-ω. In the gene cloning and recombinant plasmid construction stages, this embodiment significantly improved the efficiency of target gene acquisition and the success rate of recombinant vector construction through targeted optimization: specific primers designed based on the cat IFN-ω gene sequence can accurately bind to template cDNA, greatly reducing the interference of stray bands during PCR amplification and ensuring efficient acquisition of pure target gene PCR products; at the same time, by controlling the preset ligation time between the PCR product and the pGEM-T-Easy vector and selecting highly adaptable DH5α competent cells for transformation, the vector ligation efficiency and transformation success rate were effectively improved. Then, the recombinant expression plasmid and the pET30a vector were accurately spliced ​​by double digestion with NdeI and XhoI. Finally, a high-purity positive recombinant plasmid was obtained by identification with BL21(DE3) competent cells, laying a solid genetic foundation for subsequent efficient expression and avoiding the preparation stagnation problem caused by low gene amplification efficiency and recombinant construction failure in traditional technologies. In the recombinant expression and formulation conversion process, this embodiment achieves efficient integration from active protein to qualified formulation: BL21(DE3) competent cells are selected as the expression host, and the induction expression system under the strong promoter regulation can efficiently synthesize feline IFN-ω protein, significantly increasing the expression level of the target protein; more importantly, this embodiment breaks through the limitations of the existing technology of "emphasizing crude extraction and neglecting formulation", and directly connects to the formulation preparation process targeting the characteristics of feline interferon after induction expression. Through systematic purification, stability control and dosage form forming process, the problems of easy degradation and rapid loss of activity of interferon protein are effectively solved, ensuring that the final feline interferon formulation meets the standards for pet medical application, and realizing efficient transformation of the whole chain from gene to finished formulation; Furthermore, the preparation process involved in this embodiment is clear and highly operable, with controllable and repeatable parameters at each stage, which greatly reduces human error. This not only significantly improves the preparation efficiency of single batches of feline interferon but also meets the needs of large-scale industrial production, providing the market with sufficient and stable feline IFN-ω preparations, effectively enhancing the prevention and treatment of feline viral diseases, and possessing significant pet medical application value and industrialization prospects.

[0043] Figure 9 A flowchart of a method for detecting feline interferon according to another embodiment of the present invention is shown. This method can be executed in a computing device, wherein the computing device can be understood as a terminal with data processing capabilities, such as a mobile phone or a computer.

[0044] like Figure 9 As shown, the method begins with step S201, which includes the following: Once it is determined that the dry powder vial containing feline interferon is conveyed to the first detection area via a conveyor belt, the oscillation unit is controlled to oscillate the dry powder vial.

[0045] For example, in this embodiment, after the production of feline interferon is completed, in order to seal and store the feline interferon for subsequent sale, the production workshop generally needs to fill the feline interferon into dry powder vials. After the corresponding filling is completed, the quality of the corresponding feline interferon can be tested based on the dry powder vials in subsequent processes. It can be noted that the quality testing here may include two items: one is to determine whether the feline interferon has clumped, and the other is to determine whether the feline interferon has impurities. These two different testing items can be carried out sequentially based on an assembly line approach. The corresponding testing process involves placing vials filled with feline interferon powder onto a conveyor belt, allowing the belt to move the vials to different testing areas for different testing items. Step S201 above addresses the testing item of whether feline interferon has clumped. It should be noted that when feline interferon is filled into the vials, it is typically filled through the filling nozzle towards the center of the vial, which may result in a "less on the sides, more in the middle" distribution within the vial (e.g.,...). Figure 10 As shown in the figure, it may be difficult to accurately determine whether there is clumping in the dry powder vial under this condition. Therefore, after the dry powder vial is determined to be conveyed to the first detection area, the pre-installed oscillation unit can be controlled to oscillate the dry powder vial so that the cat interferon filled in the dry powder vial is in a more uniform state, thereby improving the detection accuracy of whether cat interferon has clumping.

[0046] For example, in this embodiment, the oscillation unit can be understood as a robotic arm, that is, the robotic arm can be used to grasp the dry powder medicine bottle and perform the corresponding oscillation operation.

[0047] Furthermore, in this embodiment, the aforementioned "determining that the dry powder vial containing feline interferon is conveyed to the first detection area via a conveyor belt" may further include the following steps: The conveying speed of the corresponding conveyor belt and the unit distance between the primary sensing unit and the secondary sensing unit set in the corresponding first detection area are determined, and the conveying time is determined based on the conveying speed and the unit distance. When it is determined that the primary gravity acquisition value output by the primary sensing unit at any acquisition moment is greater than the preset acquisition value, the secondary gravity acquisition value output by the secondary sensing unit corresponding to the transmission duration is obtained, starting from that acquisition moment. If both the primary gravity acquisition value and the secondary gravity acquisition value corresponding to the transmission duration are greater than the preset acquisition value, it is determined that the dry powder medicine bottle has been transmitted to the first detection area.

[0048] For example, in this embodiment, the process of conveying the dry powder vial to the first detection area for agitation can be implemented based on the following method steps: First, the conveying speed of the corresponding conveyor belt and the unit distance between the primary sensing unit and the secondary sensing unit set in the corresponding first detection area can be determined. Based on the conveying speed and the unit distance, the conveying time required for the dry powder medicine bottle to be conveyed from the position of the primary sensing unit to the position of the secondary sensing unit can be further determined. In order to accurately determine the position of the medicine bottle on the conveyor belt according to the time, a basis can be provided for grasping the subsequent detection timing. It can be noted that both the primary sensing unit and the secondary sensing unit can be gravity sensing units, which can be pre-installed and deployed on the conveyor belt. Then, the output of the primary sensing unit is monitored in real time. When it is determined that the primary gravity acquisition value output by the primary sensing unit at any acquisition time is greater than the preset acquisition value, it indicates that a dry powder medicine bottle may be approaching the first detection area at this time. Then, the secondary gravity acquisition value output by the secondary sensing unit corresponding to the transmission time can be obtained from the acquisition time. By monitoring the value of the secondary sensing unit within the transmission time, it can be further confirmed whether the medicine bottle has reached the first detection area as expected. Next, in response to the fact that both the primary gravity acquisition value and the secondary gravity acquisition value corresponding to the transmission duration are greater than the preset acquisition value, it indicates that within this transmission duration, the dry powder bottle has been transmitted from the primary sensing unit position to the secondary sensing unit position, and the dry powder bottle is simultaneously located on both the primary and secondary sensing units. At this point, it is determined that the dry powder bottle has been transmitted to the first detection area, and then the oscillation unit is controlled to oscillate the dry powder bottle. It can be noted that, in this embodiment, through the dual verification of the primary and secondary sensing units, misjudgment can be effectively avoided, ensuring that the oscillation operation is performed when the dry powder bottle accurately arrives at the first detection area. This provides a reliable premise for subsequently determining the first detection value of the block dimension based on the oscillated image, ensuring the accuracy and effectiveness of quality detection.

[0049] Similarly, in this embodiment, the above-mentioned "controlling the oscillation unit to oscillate the dry powder medicine bottle" may further include the following steps: The first acquisition unit corresponding to the first detection area is controlled to acquire images of the dry powder medicine bottle, and the pixels of each medicine bottle that make up the cat interferon are determined based on the obtained medicine bottle image. Based on the coordinate processing of the medicine bottle image, the image coordinate points corresponding to each medicine bottle pixel are obtained, and the medicine bottle coordinate points corresponding to the horizontal maximum value and the horizontal minimum value are determined as the first side coordinate point and the second side coordinate point. The first side coordinate point and the second side coordinate point are calculated based on the difference of the horizontal coordinate value, and the medicine bottle coordinate point with the same horizontal value as the obtained horizontal difference is determined as the center coordinate point; The difference between the center coordinate point and the first side coordinate point and the second side coordinate point is calculated based on the vertical coordinate. The average of the first vertical difference and the second vertical difference is calculated to obtain the vertical average. The oscillation adjustment coefficient is obtained by weighted summation of the horizontal difference and the vertical mean. The reference oscillation parameters of the corresponding oscillation unit are updated based on the oscillation adjustment coefficient, and the oscillation unit is controlled to oscillate the dry powder medicine bottle based on the obtained current oscillation parameters.

[0050] For example, in this embodiment, the shaking operation on the dry powder medicine bottle can be specifically implemented based on the following method steps: First, when the dry powder vial arrives at the first detection area, the server can control the first acquisition unit corresponding to the first detection area to acquire images of the dry powder vial. Through image acquisition, visual information of the corresponding dry powder vial can be obtained, and the pixel points of each vial that make up the cat interferon can be determined based on the obtained vial image, providing an image basis for subsequent accurate analysis of the vial. Then, based on the coordinate processing of the medicine bottle image, the pixels in the image are transformed into coordinate points with clear position information to obtain the image coordinate points corresponding to each medicine bottle pixel. Furthermore, the medicine bottle coordinate points corresponding to the horizontal maximum and horizontal minimum values ​​are determined as the first side coordinate point and the second side coordinate point. These two coordinate points can determine the horizontal boundary position of cat interferon in the dry powder medicine bottle. Next, the difference between the first side coordinate point and the second side coordinate point is calculated based on the horizontal coordinate value. The obtained horizontal difference reflects the size of the dry powder medicine bottle in the horizontal direction. The medicine bottle coordinate point with the same horizontal value as the obtained horizontal difference is determined as the center coordinate point. This center coordinate point can be used as a reference for the horizontal center position of the dry powder medicine bottle. Subsequently, the difference between the central coordinate point and the first side coordinate point and the second side coordinate point is calculated based on the longitudinal coordinate to obtain the first longitudinal difference and the second longitudinal difference. These two differences reflect the offset of cat interferon relative to the center in the longitudinal direction. The average of the first longitudinal difference and the second longitudinal difference is calculated to obtain the longitudinal average. This longitudinal average can comprehensively reflect the characteristics of cat interferon in the longitudinal direction. Then, the horizontal difference and the vertical mean are weighted and summed. Considering the different degrees of influence of horizontal and vertical features on the oscillation operation, the oscillation adjustment coefficient is obtained by weighted summation. This coefficient can adjust the oscillation parameters according to the specific shape characteristics of the medicine bottle. Finally, based on the oscillation adjustment coefficient, the reference oscillation parameters of the corresponding oscillation unit are updated to make the oscillation parameters more suitable for the shape characteristics of the current medicine bottle. Based on the obtained current oscillation parameters, the oscillation unit is controlled to oscillate the dry powder medicine bottle. Through this adjustment of oscillation parameters based on the image features of the medicine bottle, the cat interferon in the medicine bottle can be more effectively kept in a loose and uniform state, which provides a guarantee for the subsequent accurate detection of quality problems such as clumping in the medicine bottle, and improves the accuracy and reliability of quality detection.

[0051] Furthermore, in this embodiment, the aforementioned "updating the reference oscillation parameters corresponding to the oscillation unit based on the oscillation adjustment coefficient, and controlling the oscillation unit to perform oscillation operation on the dry powder medicine bottle based on the obtained current oscillation parameters" may further include the following steps: When the oscillation adjustment coefficient is greater than the retrieved oscillation adjustment threshold, an adjustment multiple corresponding to the oscillation adjustment threshold is determined based on the oscillation adjustment coefficient. The reference oscillation rate and reference oscillation time are updated based on the adjustment factor to obtain the current oscillation rate and current oscillation time. The oscillation unit is controlled to perform a first oscillation operation in the horizontal direction corresponding to the current oscillation time based on the current oscillation rate; Upon completion of the first oscillation operation, the oscillation unit is controlled to perform a second oscillation operation in the vertical direction corresponding to the number of continuous reference oscillations on the dry powder vial.

[0052] For example, in this embodiment, the oscillation operation based on the current oscillation parameters obtained by updating the reference oscillation parameters can be implemented using the following method steps: First, if the oscillation adjustment coefficient is greater than the retrieved oscillation adjustment threshold, it indicates that the morphological characteristics of the feline interferon in the current dry powder vial require stronger oscillation to achieve effective loosening. At this time, the adjustment multiple corresponding to the oscillation adjustment threshold can be determined based on the numerical relationship between the oscillation adjustment coefficient and the oscillation adjustment threshold. Furthermore, the adjustment multiple can be used to quantify the adjustment range of the oscillation parameters, providing a basis for precise control of the oscillation intensity. Then, based on the adjustment factor, the reference oscillation rate and reference oscillation time are updated. That is, by adjusting the rate and time, the energy output of the oscillation operation is matched with the real-time state of the cat interferon in the dry powder vial, and the current oscillation rate and current oscillation time are obtained. Next, the oscillation unit is controlled to perform a first oscillation operation in the horizontal direction corresponding to the current oscillation time based on the current oscillation rate. The horizontal oscillation can make the cat interferon present a uniform distribution on the cross-section of the bottle as much as possible, so as to provide a clear detection basis for subsequent image detection of the block dimension. Finally, after the first oscillation operation is completed, the oscillation unit can be controlled again to perform a second oscillation operation in the vertical direction corresponding to the reference number of oscillations on the dry powder vial. The vertical oscillation can further eliminate the accumulation of feline interferon in the longitudinal direction. The standardization of the oscillation process is ensured by a fixed number of reference operations, avoiding excessive or insufficient oscillation from affecting the accuracy of the detection results.

[0053] By implementing the aforementioned phased and directional dynamic adjustment mechanism for oscillation parameters, personalized oscillation processing can be achieved for the actual shape characteristics of different vials, while the consistency of the detection process can be ensured through standardized vertical oscillation steps. This improves the reliability of detecting clumping in cat interferon dry powder vials, ensures that the oscillation operation effectively assists subsequent image analysis, and enhances the accuracy and stability of quality detection.

[0054] Step S202 includes the following: Upon completion of the oscillation operation, the first acquisition unit corresponding to the first detection area acquires an image of the dry powder medicine bottle and determines a first detection value for the corresponding agglomeration dimension based on the obtained first detection image.

[0055] For example, in this embodiment, after the oscillation unit completes the oscillation operation on the dry powder medicine bottle, the first acquisition unit corresponding to the first detection area can be controlled to acquire images of the dry powder medicine bottle to obtain the first detection image of the corresponding dry powder medicine bottle. Since the first detection image can reflect the actual state of the cat interferon filled in the dry powder medicine bottle, the first detection value of the corresponding agglomeration dimension can be further determined based on the first detection image. It can be explained that the first acquisition unit can be understood as a camera, which can acquire images of the dry powder medicine bottle from the corresponding frontal view.

[0056] Furthermore, in this embodiment, the aforementioned "responding to the completion of the oscillation operation, controlling the first acquisition unit corresponding to the first detection area to acquire an image of the dry powder medicine bottle, and determining the first detection value corresponding to the clumping dimension based on the obtained first detection image" may further include the following steps: The oscillation operation is completed in response to control the first acquisition unit to acquire images of the dry powder medicine bottle, and the first pixel points that make up the cat interferon are determined based on the first acquired image. Based on the coordinate processing of the first acquired image, the first coordinate point corresponding to each first pixel is obtained, and the horizontal coordinate value corresponding to each first pixel is used to form a horizontal numerical range. All first coordinate points that have the same horizontal value as each horizontal coordinate value in the horizontal data interval are determined as horizontal filtering groups, and the first coordinate point located at the corresponding vertical maximum value of each horizontal filtering group is determined as surface coordinate point. For each surface coordinate point in an adjacent position, the difference based on the longitudinal coordinate value is calculated, and the first detection value of the corresponding block dimension is determined based on the obtained absolute difference in each longitudinal direction.

[0057] For example, in this embodiment, the specific steps for determining the first detection value of the corresponding block dimension based on the first detection image can be described as follows: First, after the oscillation operation is completed, the first acquisition unit is controlled to acquire images of the dry powder medicine bottle. The distribution state of the cat interferon in the medicine bottle after oscillation is obtained through image acquisition. Based on the obtained first acquisition image, the first pixel points that make up the cat interferon are determined, providing basic data for subsequent analysis of the distribution characteristics of cat interferon. Then, based on the coordinate processing of the first acquired image, that is, the pixel points in the image are converted into coordinate points with clear position information, so as to obtain the first coordinate point corresponding to each first pixel point, and further, the horizontal coordinate values ​​corresponding to each first coordinate point are combined into a horizontal numerical range. It can be explained that the horizontal numerical range can reflect the distribution range of cat interferon in the dry powder bottle in the horizontal direction. Next, all first coordinate points that have the same horizontal value as each horizontal coordinate value in the horizontal data interval are determined as horizontal filtering groups. Each horizontal filtering group contains all pixel information at the same horizontal position. The first coordinate point located at the vertical maximum value corresponding to each horizontal filtering group is determined as a surface coordinate point. These surface coordinate points constitute the outline of the surface of cat interferon and can be used to analyze the surface undulation of cat interferon. Finally, the difference between the longitudinal coordinates of each adjacent surface coordinate point can be calculated. The longitudinal difference between adjacent surface coordinate points reflects the local height change of the surface of cat interferon. Based on the obtained longitudinal absolute difference, the first detection value of the corresponding clumping dimension is determined. By analyzing these longitudinal absolute differences, clumping that may exist in cat interferon can be detected, because the surface height change of the clumping area is usually greater than that of the normally dispersed dry powder area. Through this image analysis-based clumping detection method, the morphological characteristics of the surface of cat interferon can be accurately obtained, providing a reliable basis for judging whether there are clumping in the dry powder vial and the severity of the clumping, thereby effectively ensuring the quality of the dry powder vial containing cat interferon.

[0058] Furthermore, in this embodiment, the aforementioned "determining the first detection value corresponding to the block dimension based on the obtained longitudinal differences" may further include the following steps: The surface coordinate points corresponding to each vertical difference are sorted in descending order of horizontal coordinate values ​​to obtain a coordinate sequence. If any longitudinal difference is greater than a preset difference, the next position of the two surface coordinate points corresponding to the longitudinal difference is determined to have agglomeration properties based on the coordinate sequence. Based on the coordinate sequence, all surface coordinate points of the corresponding block attributes that are arranged in a continuous manner are aggregated into the same block determination group, and the longitudinal coordinate value of the corresponding longitudinal maximum value is determined as the block height value based on all surface coordinate points located in the same block determination group. The height evaluation value is determined based on the block height value of each block group, and the quantity evaluation value is determined based on the number of blocks in all block groups. The height evaluation value and the quantity evaluation value are weighted and summed to obtain the first detection value.

[0059] For example, in this embodiment, the specific steps for determining the first detection value based on the longitudinal difference can be described as follows: First, the surface coordinate points corresponding to each vertical difference can be sorted from largest to smallest based on the horizontal coordinate value to obtain a coordinate sequence. By sorting, the surface coordinate points can be arranged in an orderly manner according to the horizontal position, which is convenient for subsequent analysis of the relationship between adjacent points. Then, if any vertical difference is greater than a preset difference, it may indicate that there may be a blockage at that location. Based on the coordinate sequence, the second-ranked of the two surface coordinate points corresponding to the vertical difference can be identified as having a blockage attribute, so as to filter out possible blockage areas by setting a threshold. Next, based on the coordinate sequence, all surface coordinate points of the corresponding block attributes that are arranged in a continuous manner are summarized into the same block determination group. Since the coordinate points arranged in a continuous manner are more likely to belong to the same block, the longitudinal coordinate value of the corresponding longitudinal maximum value can be determined as the block height value based on all surface coordinate points located in the same block determination group. This height value can reflect the size of the block in the longitudinal direction. Subsequently, a height evaluation value is determined based on the clump height value corresponding to each clump determination group. The height evaluation value can quantify the severity of a single clump. A quantity evaluation value is determined based on the number of clumps corresponding to all clump determination groups. The quantity evaluation value can reflect the overall distribution of clumps in the dry powder vial. Finally, the height evaluation value and the quantity evaluation value are weighted and summed. Considering the different degrees of influence of the clump height and quantity on the quality of the feline interferon in the dry powder vial, the first detection value is obtained by weighted summation. This first detection value comprehensively reflects the severity of clumping in the dry powder vial, providing an important basis for judging whether the quality of feline interferon meets the standard, and improving the accuracy and reliability of quality detection.

[0060] It can be explained that the larger the block height value and the more blocks there are, the smaller the corresponding height evaluation value and quantity evaluation value, which in turn leads to a smaller first detection value.

[0061] Step S203 includes the following: When it is determined that the dry powder medicine bottle has been transferred from the first detection area to the second detection area, the tilting unit is controlled to tilt the dry powder medicine bottle.

[0062] For example, in this embodiment, based on the foregoing, steps S201 and S202 are both technical solutions executed to determine whether cat interferon has agglomerated. After completing this detection item, the detection item to determine whether cat interferon has impurities can be performed immediately. It can be noted that, for the detection item of impurities, since cat interferon filled in dry powder bottles is generally presented in the form of lyophilized powder, when impurities are present, in some cases, the impurities may be buried in the cat interferon, making it impossible to determine the impurities based on vision. In this case, the tilting unit corresponding to the second detection area can be controlled to tilt the dry powder bottle, so that the cat interferon in the dry powder bottle can flow under the action of gravity, helping the impurities buried in the cat interferon to become exposed, so that the impurities can be determined based on vision, thereby improving the detection accuracy of the detection item of whether cat interferon has impurities.

[0063] For example, in this embodiment, the tilting unit can also be understood as a robotic arm, that is, the robotic arm can be used to grasp the dry powder medicine bottle and perform the corresponding tilting operation.

[0064] Furthermore, in this embodiment, the aforementioned "when it is determined that the dry powder medicine bottle has been transferred from the first detection area to the second detection area, control the tilting unit to tilt the dry powder medicine bottle" may further include the following steps: Retrieve the tilt specification table, which includes multiple preset tilt angles; When it is determined that the dry powder medicine bottle is transferred from the first detection area to the second detection area, the tilt control unit tilts the dry powder medicine bottle based on each preset tilt angle located in the tilt specification table.

[0065] For example, in this embodiment, the tilting operation of the dry powder medicine bottle can be specifically implemented based on the following method steps: First, a pre-stored tilt specification table can be retrieved. The tilt specification table includes multiple preset tilt angles, which are preset based on the physical characteristics of cat interferon dry powder and the actual needs of quality testing, providing standard parameter basis for subsequent tilting operations. Finally, when it is determined that the dry powder vial has been transferred from the first detection area to the second detection area, it indicates that the vial has completed the preliminary detection and is ready to proceed to the next detection item. At this time, the server can control the tilting unit to tilt the dry powder vial based on each preset tilt angle in the tilt specification table. By tilting the vial at multiple different angles, the cat interferon inside the vial can be made to flow and shift to different degrees under the action of gravity, so as to more comprehensively expose any possible impurities and provide richer information for subsequent image analysis based on different tilt angles, thereby improving the accuracy and reliability of quality detection.

[0066] Step S204 includes the following: The second acquisition unit corresponding to the second detection area, in response to the tilt operation control, acquires an image of the dry powder vial and determines a second detection value for the corresponding impurity dimension based on the obtained second detection image.

[0067] For example, in this embodiment, after the tilting unit completes the tilting operation on the dry powder medicine bottle, the second acquisition unit corresponding to the second detection area can be controlled to acquire images of the dry powder medicine bottle to obtain the second detection image of the dry powder medicine bottle, and further determine the second detection value of the corresponding impurity dimension based on the second detection image; it can be explained that the second acquisition unit can also be understood as a camera, which can acquire images of the dry powder medicine bottle from the corresponding frontal view.

[0068] Furthermore, in this embodiment, the aforementioned "responding to the tilt operation control of the second acquisition unit corresponding to the second detection area to acquire an image of the dry powder vial, and determining the second detection value corresponding to the impurity dimension based on the obtained second detection image" may further include the following steps: The response is to complete the tilting operation based on any preset tilt angle, control the second acquisition unit corresponding to the second detection area to acquire the image of the dry powder medicine bottle, and perform binarization processing on the obtained second detection image to obtain a binarized image that includes at least the second pixel point corresponding to the dry powder pixel value; If a noise pixel with a corresponding noise pixel value exists in the binarized image, the preset tilt angle corresponding to the second detection image is determined as the impurity identification angle, and the number of noise pixels corresponding to the noise pixel is obtained. The response completes the tilting operation based on all preset tilt angles, determines the maximum number of noise pixels based on all impurity identification angles, and determines the second detection value of the corresponding impurity dimension based on the number of noise pixels.

[0069] For example, in this embodiment, determining the second detection value corresponding to the impurity dimension based on the second detection image can be specifically implemented based on the following method steps: First, the response indicates that the dry powder bottle is in a specific tilt state when the tilting operation is completed based on any preset tilt angle. The server can then control the second acquisition unit corresponding to the second detection area to acquire images of the dry powder bottle. The distribution of cat interferon in the dry powder bottle under the tilt state is obtained through image acquisition. The obtained second detection image is then binarized to classify the pixels in the second detection image into different categories, resulting in a binarized image that includes at least the second pixel value corresponding to the dry powder. Binarization can simplify the image analysis process and highlight the morphological features of the dry powder. Then, in response to the existence of noise pixels with corresponding noise pixel values ​​in the binarized image, these noise pixels may represent impurities present in the medicine bottle. Therefore, the preset tilt angle corresponding to the second detection image can be determined as the impurity identification angle. That is, the second detection image obtained under the preset tilt angle is more conducive to identifying impurities, and at the same time, the number of noise pixels corresponding to the noise pixels can be obtained. It can be explained that the number of noise pixels can reflect the size of the impurities. Finally, the response completes the tilting operation based on all preset tilt angles, indicating that the dry powder vial has been comprehensively inspected at multiple angles. At this point, the maximum number of noise pixels can be determined based on all impurity identification angles. The maximum number is selected to ensure that the most severe impurities are detected, and a second detection value for the corresponding impurity dimension is determined based on this number of noise pixels. It can be noted that this second detection value can accurately reflect the severity of impurities in the dry powder vial. By acquiring and analyzing images of the dry powder vial at multiple preset tilt angles, the impurities in the vial can be detected more comprehensively and accurately, improving the reliability of quality inspection and ensuring the quality of dry powder vials filled with cat interferon.

[0070] It can be noted that the more noisy pixels there are, the smaller the corresponding second detection value should be.

[0071] In addition, in this embodiment, the tilting operation of the dry powder medicine bottle is based on each preset tilt angle in the tilt specification table. The preset tilt angles may include 5 degrees, 10 degrees, 15 degrees, 20 degrees, etc. Each preset tilt angle may change the position of the dry powder medicine bottle from buried to exposed. In order to improve the corresponding detection efficiency, this embodiment may further include the following steps: Obtain the number of angle detections for each preset tilt angle located in the tilt specification table; When any preset tilt angle is determined as the impurity identification angle, the angle detection count for that preset tilt angle is reset to zero. If any preset tilt angle is not determined as an impurity identification angle, the angle detection count for that preset tilt angle is accumulated to obtain the updated angle detection count. If it is determined that the number of times any updated angle is detected is greater than the preset number of deletions, the preset tilt angle corresponding to the number of times the angle is detected will be removed from the tilt specification table.

[0072] For example, in this embodiment, updating the tilt specification table can be achieved based on the following method steps: First, the number of angle detections for each preset tilt angle in the tilt specification table can be obtained. It can be noted that the number of angle detections records the usage of each preset tilt angle in the historical detection process. Then, when any preset tilt angle is determined as the impurity identification angle, it indicates that the impurity has been successfully identified in the current detection. The angle detection count for the preset tilt angle is then cleared to start counting again, so as to avoid the impact of excessive historical cumulative counts on subsequent judgments. Next, if any preset tilt angle is not determined as an impurity identification angle, it indicates that the angle has failed to effectively identify impurities in the current detection. The angle detection count for the preset tilt angle is accumulated to obtain the updated angle detection count. The effectiveness of the angle can be evaluated by accumulating the number of unidentified impurities. Finally, if it is determined that the number of times any updated angle is detected is greater than the preset deletion number, it indicates that the preset tilt angle has not played an effective role in multiple detections. The preset tilt angle corresponding to the number of times the angle is detected will be removed from the tilt specification table. Through this dynamic management mechanism, inefficient preset tilt angles can be eliminated in a timely manner, the tilt detection process can be optimized, unnecessary detection steps can be reduced, the efficiency and accuracy of quality detection can be improved, and the detection cost can be reduced, ensuring the efficient operation of quality detection for dry powder vials of cat interferon.

[0073] Step S205 may also include the following: The quality status of the corresponding dry powder medicine bottle is determined based on the first detection value and the second detection value.

[0074] For example, in this embodiment, after obtaining the first detection value corresponding to the clumping dimension and the second detection value corresponding to the impurity dimension, the quality status of the corresponding dry powder medicine bottle can be determined based on the two. The quality status can include a defective status and a qualified status. When the corresponding status is defective, it means that the dry powder medicine bottle needs to be rejected and should not be used in the market. When the corresponding status is qualified, it means that the dry powder medicine bottle can be used in the market.

[0075] Furthermore, in this embodiment, the aforementioned "determining the quality status of the corresponding dry powder medicine bottle based on the first detection value and the second detection value" may further include the following steps: A testing notification page is established based on the batch of the dry powder medicine bottles, wherein the testing notification page includes a clumping detection slot and an impurity detection slot; Fill the agglomeration detection slot and the impurity detection slot with the first detection image and the second detection image, respectively; When the first detection value is less than the preset agglomeration threshold, the quality status of the corresponding dry powder medicine bottle is determined to be a defective state, and the groove contour of the agglomeration detection groove is pixel-rendered according to the first pixel value. When the second detection value is less than the preset impurity threshold, the quality status of the corresponding dry powder medicine bottle is determined to be a defective state, and the groove outline of the impurity detection groove is pixel rendered according to the second pixel value. When the first detection value and the second detection value are respectively greater than or equal to the preset block threshold, the first detection value and the second detection value are weighted and summed to obtain a comprehensive evaluation value. When the comprehensive evaluation value is greater than or equal to the preset comprehensive threshold, the quality status of the corresponding dry powder medicine bottle is determined to be qualified, and the groove contours of the agglomeration detection groove and the impurity detection groove are respectively rendered with the corresponding third pixel value. Otherwise, it is determined to be defective, and the corresponding fourth pixel value is rendered.

[0076] For example, in this embodiment, the determination of the quality status of the corresponding dry powder medicine bottle can be specifically achieved based on the following method steps: First, a testing notification page is established based on the batch of the dry powder medicine bottles. The testing notification page is equipped with a clumping detection slot and an impurity detection slot, which facilitates the systematic management and visual presentation of the quality of medicine bottles by batch. Then, the first detection image and the second detection image are filled into the agglomeration detection slot and the impurity detection slot respectively, providing a visual basis for quality judgment through intuitive image information; In one case, if the first detection value is less than a preset clumping threshold, it indicates that the clumping situation in the medicine bottle exceeds the acceptable range. The quality status of the corresponding dry powder medicine bottle is determined to be a defective state, and the groove contour of the clumping detection groove is pixel-rendered according to the first pixel value, so as to achieve rapid identification of the defective state through color marking of specific pixel values. In another case, if the second detection value is less than the preset impurity threshold, it indicates that the impurity content in the medicine bottle exceeds the standard. The quality status of the corresponding dry powder medicine bottle is determined to be a defective state, and the outline of the impurity detection slot is pixel-rendered according to the second pixel value to highlight the quality problem of the impurity dimension with differentiated visual identification. In another scenario, when the first detection value and the second detection value are greater than or equal to the preset clumping threshold and the preset impurity threshold, respectively, it indicates that neither of the two key indicators directly triggers a defect determination. In this case, the first detection value and the second detection value are weighted and summed to obtain a comprehensive evaluation value. By comprehensively considering the detection results of clumping and impurities through weighted calculation, the quality judgment becomes more comprehensive. Furthermore, in response to the comprehensive evaluation value being greater than or equal to the preset comprehensive threshold, the quality status of the corresponding dry powder medicine bottle is determined to be qualified, and the slot contours of the clumping detection slot and the impurity detection slot are respectively rendered with the corresponding third pixel value. Conversely, if they are not, it is determined to be a defective state, and the corresponding fourth pixel value is rendered.

[0077] Through the above process, a full-chain quality status determination mechanism is realized, from image visualization to multi-dimensional indicator judgment and result visualization. This not only meets the traceability requirements of batch management, but also improves the readability and processing efficiency of test results through pixel rendering technology, ensuring the accuracy and efficiency of quality testing of cat interferon dry powder vials.

[0078] In summary, according to the solution of this embodiment, this embodiment significantly improves the efficiency and accuracy of quality inspection of dry powder vials filled with feline interferon; In the clumping detection stage, this embodiment can accurately determine whether the medicine bottle has reached the first monitoring area through the linkage design of the conveyor belt speed and the sensing unit, avoiding detection omissions or misoperations caused by position misjudgment. The oscillation unit can dynamically adjust the oscillation parameters based on the coordinate analysis of the medicine bottle image. The oscillation adjustment coefficient is generated by the weighted calculation of the horizontal difference and the vertical mean, realizing personalized oscillation processing for medicine bottles of different shapes. It can effectively loosen the cat interferon evenly, making the clumping easier to expose in subsequent image acquisition, and significantly improving the sensitivity of clumping detection. In the impurity detection stage, the cat interferon can be fully flowed under gravity by multi-angle operation in the preset tilt specification table. Impurities (such as foreign particles) are more easily separated from normal dry powder due to differences in physical properties. Combined with binarized image analysis and noise pixel recognition, the impurity content can be accurately quantified. At the same time, inefficient preset tilt angles can be removed based on the number of angle detections for each preset tilt angle, continuously optimizing the detection process, reducing redundant operations, and improving detection efficiency. In addition, the comprehensive judgment of quality status integrates data from both agglomeration and impurities. A comprehensive evaluation value is generated through weighted summation, and combined with a visual detection notification page and pixel rendering, rapid identification of detection results is achieved. It can be noted that this embodiment not only provides real-time feedback for production process control, but also reduces human interpretation errors through standardized processes, ensuring that unqualified products can be intercepted in a timely manner, effectively guaranteeing the production quality of feline interferon and improving the level of automated detection.

[0079] Another embodiment of the present invention provides a quality detection system for feline interferon. Figure 11 Its corresponding system block diagram, such as Figure 11 As shown, the system includes: The oscillation operation module is configured to control the oscillation unit to oscillate the dry powder vial when it is determined that the dry powder vial containing cat interferon is conveyed to the first detection area via a conveyor belt. The clumping detection module is configured to respond to the completion of the oscillation operation by controlling the first acquisition unit corresponding to the first detection area to acquire an image of the dry powder medicine bottle, and to determine a first detection value corresponding to the clumping dimension based on the obtained first detection image. The tilting operation module is configured to control the tilting unit to tilt the dry powder medicine bottle when it is determined that the dry powder medicine bottle is transferred from the first detection area to the second detection area. The impurity detection module is configured to respond to a tilt operation by controlling the second acquisition unit corresponding to the second detection area to acquire an image of the dry powder vial, and to determine a second detection value for the corresponding impurity dimension based on the obtained second detection image. The status determination module is configured to determine the quality status of the corresponding dry powder medicine bottle based on the first detection value and the second detection value.

[0080] In the specification provided herein, the algorithms and displays are not inherently related to any particular computer, virtual system, or other device. Various general-purpose systems can also be used with the examples of this invention. The required structure for constructing such systems is apparent from the above description. Furthermore, this invention is not directed to any particular programming language. It should be understood that the contents of the invention described herein can be implemented using various programming languages, and the above description of specific languages ​​is for the purpose of disclosing preferred embodiments of the invention.

[0081] Numerous specific details are set forth in the specification provided herein. However, it will be understood that embodiments of the invention may be practiced without these specific details. In some instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this specification.

[0082] Similarly, it should be understood that, in order to streamline this disclosure and aid in understanding one or more of the various aspects of the invention, in the description of exemplary embodiments of the invention above, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof.

[0083] Those skilled in the art will understand that modules, units, or components of the devices disclosed in the examples herein can be arranged in the devices described in this embodiment, or alternatively, can be located in one or more devices different from the devices in this example. The modules in the foregoing examples can be combined into a single module or, in addition, can be divided into multiple sub-modules.

[0084] Those skilled in the art will understand that the modules in the device of the embodiment can be adaptively changed and placed in one or more devices different from that embodiment. Modules, units, or components in the embodiment can be combined into a single module, unit, or component, and further, they can be divided into multiple sub-modules, sub-units, or sub-components.

[0085] Furthermore, those skilled in the art will understand that although some embodiments described herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of the invention and form different embodiments.

[0086] Furthermore, some of the embodiments described herein are methods or combinations of method elements that can be implemented by a processor of a computer system or by other means of performing the functions. Therefore, a processor having the necessary instructions for implementing the methods or method elements forms means for implementing the methods or method elements. Furthermore, the elements described herein in the apparatus embodiments are examples of means for implementing the functions performed by elements for the purposes of carrying out the invention.

[0087] As used herein, unless otherwise specified, the use of ordinal numbers such as “first,” “second,” “third,” etc., to describe ordinary objects merely indicates different instances of similar objects and is not intended to imply that the objects being described must have a given order in time, space, ordering, or any other manner.

[0088] Although the invention has been described with respect to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and edibility purposes, and not for the purpose of explaining or limiting the subject matter of the invention.

Claims

1. A method for preparing a recombinant feline-ω interferon antiviral agent, characterized in that, Includes the following steps: Total RNA extracted from the corresponding cat spleen was reverse transcribed to obtain cDNA; Specific primers were determined based on the feline IFN-ω gene sequence, and the cDNA was used as a template to perform PCR amplification on the feline IFN-ω gene sequence using the specific primers to obtain PCR products. The PCR product was ligated to a universal vector for a predetermined duration, and then transferred into DH5α competent cells after the ligation was completed. Using specific primers, positive clone plasmids were extracted from the DH5α competent cells to obtain recombinant expression plasmids. The recombinant expression plasmids and pET30a vector were double-digested with NdeI and XhoI. The recovered target fragments were then transformed into BL21(DE3) competent cells to identify positive recombinant plasmids containing the target gene. The positive recombinant plasmid containing the target gene was transformed into BL21(DE3) competent cells for colony culture, and the resulting cultured cells were induced to express the gene. After the induction expression was completed, the cultured bacterial cells were purified and the corresponding feline interferon was prepared.

2. The preparation method according to claim 1, characterized in that, Specific primers were determined based on the feline IFN-ω gene sequence, including: The obtained feline IFN-ω gene sequence was modified by removing the corresponding signal peptide and adding the start codon ATG at the 5' end. After adding the corresponding start codon ATG, restriction enzyme sites Nde and XhoI are introduced at the 5' end of the primers to obtain specific primers, wherein the specific primers include the upstream primer FeIFN-ωF and the downstream primer FeIFN-ωR.

3. The preparation method according to claim 1, characterized in that, The PCR product is ligated to a universal vector for a predetermined duration, and then transformed into DH5α competent cells after the ligation is complete. This includes: The PCR product was purified and recovered, and the purified and recovered PCR product was ligated with a universal vector at 16°C for 4 hours. After the ligation operation is completed, the ligated PCR product and universal vector are transformed into DH5α competent cells.

4. The preparation method according to claim 3, characterized in that, Using specific primers, positive clone plasmids were extracted from the DH5α competent cells and verified to be correct, resulting in recombinant expression plasmids, including: DH5α competent cells were seeded in liquid culture medium and cultured with shaking at 37°C and 200 r / min. Positive clone plasmids were extracted and verified from DH5α competent cells after oscillation culture using upstream primer FeIFN-ωF and downstream primer FeIFN-ωR.

5. The preparation method according to claim 1, characterized in that, The purification and formulation preparation of the cultured bacterial cells corresponding to feline interferon includes: The cultured bacterial cells were disrupted, and the disrupted bacterial cells were washed with inclusion bodies using urea and Triton solution to obtain lysis products. The lysis products were separated by chromatography, and the proteins obtained after chromatography were replaced with refolding buffer using G-25 packing material. The refolding buffer solution was stirred within a temperature range of 2-8°C and a time range of 8-24 hours, and then filtered using Sephadex G-25 gel after the stirring operation was completed. The obtained filtered product was placed in PBS buffer, and the corresponding feline interferon was prepared based on the obtained FeIFN-ω protein stock solution.

6. The preparation method according to claim 5, characterized in that, The preparation of corresponding feline interferon formulations based on the obtained FeIFN-ω protein stock solution includes: Phosphate, sucrose, and amino acids were formulated into a freeze-drying protectant. FeIFN-ω protein stock solution was added to the freeze-drying protectant and mixed evenly. The resulting feline interferon was then dispensed and freeze-dried.

7. A method for detecting feline interferon prepared based on the preparation method of the recombinant feline-ω interferon antiviral agent according to any one of claims 1-6, characterized in that, Includes the following steps: Once it is determined that the dry powder vial containing feline interferon is conveyed to the first detection area via a conveyor belt, the oscillation unit is controlled to oscillate the dry powder vial. The oscillation operation control unit corresponding to the first detection area performs image acquisition on the dry powder medicine bottle, and determines the first detection value of the corresponding agglomeration dimension based on the obtained first detection image. When it is determined that the dry powder medicine bottle is transferred from the first detection area to the second detection area, the tilting unit is controlled to tilt the dry powder medicine bottle. The response completes the tilt operation control, the second acquisition unit corresponding to the second detection area performs image acquisition on the dry powder medicine bottle, and determines the second detection value of the corresponding impurity dimension based on the obtained second detection image; The quality status of the corresponding dry powder medicine bottle is determined based on the first detection value and the second detection value.

8. The detection method according to claim 1, characterized in that, The process involves ensuring that vials containing feline interferon powder are conveyed to the first detection area via a conveyor belt, including: The conveying speed of the corresponding conveyor belt and the unit distance between the primary sensing unit and the secondary sensing unit set in the corresponding first detection area are determined, and the conveying time is determined based on the conveying speed and the unit distance. When it is determined that the primary gravity acquisition value output by the primary sensing unit at any acquisition moment is greater than the preset acquisition value, the secondary gravity acquisition value output by the secondary sensing unit corresponding to the transmission duration is obtained, starting from that acquisition moment. If both the primary gravity acquisition value and the secondary gravity acquisition value corresponding to the transmission duration are greater than the preset acquisition value, it is determined that the dry powder medicine bottle has been transmitted to the first detection area.

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