Recombinant expression cat interferon-omega gene as well as preparation method and application thereof

Abstract

The invention relates to a recombinant expression cat interferon-omega gene and a preparation method and application thereof, the nucleotide sequence of the cat interferon-omega gene is as shown in SEQ ID NO.1, and the cat interferon-omega gene is obtained by introducing an Fc fusion fragment to the C terminal and / or N terminal of the natural gene sequence of the cat interferon-omega. XTEN or PAS is introduced to prolong a peptide fragment, and original glycosylation modification sites on a natural sequence are reserved. The method comprises the following steps: cloning a cat interferon-omega gene into an expression vector containing double promoters, further transfecting into a cell, carrying out stable cloning and screening, establishing a high-expression cell strain, and carrying out fermentation culture in a bioreactor. According to the invention, the cat interferon-omega gene is subjected to multiple modification and is efficiently expressed in a CHO-K1GS system, so that the protein yield, the stability and the half-life period are remarkably improved; the obtained fusion protein is high in purity and strong in activity, can obviously enhance immune response and protection effect when being matched with cat vaccines, and is good in safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals and veterinary immunology, in particular to a recombinantly expressed feline interferon-omega gene and its preparation method and application. BACKGROUND

[0002] Feline interferon-omega (IFN-omega) is a natural antiviral protein produced by felines, which has important application value in the field of feline infectious disease prevention and control due to its broad-spectrum antiviral activity and immunomodulatory function. Currently, commercially available feline IFN-omega products are mostly prepared through eukaryotic or prokaryotic expression systems, but there are generally problems such as low expression efficiency, unstable protein biological activity, short in vivo half-life, etc. Among them, the short half-life leads to frequent administration in clinical application, which not only increases the use cost, but also reduces the compliance of pet owners, limiting its practical application effect in feline disease prevention and control.

[0003] With the development of biological engineering technology, significant progress has been made in protein expression systems and long-acting modification methods. CHO cells, as a mature industrial expression platform, have good protein folding and post-translational modification capabilities; through the introduction of expression enhancers, signal peptide engineering, glycosylation optimization and fusion peptide segments, etc., the yield, biological activity and in vivo stability of recombinant proteins can be effectively improved. However, there are still deficiencies in the existing technology in terms of high-efficiency expression, long-acting modification and synergistic application with vaccines of feline IFN-omega.

[0004] Patent CN113337472A provides a recombinant CHO DG44 cell line for high-efficiency and stable expression of feline interferon-omega 2. By integrating the FeIFN-omega 2 gene at a specific site into the high-transcriptional activity region of the short arm of chromosome 1 of CHO cells, the expression efficiency and stability of the exogenous gene are improved. At the same time, using fluorescence in situ hybridization technology, a homozygous cell clone without chromosomal gaps is obtained, so that the obtained cell line can still maintain high-level stable expression after 50 generations of continuous passage, and the highest yield can reach 1.269 g / L. The antiviral specific activity of the purified protein reaches 6.3 x 10^8 U / mg, which is significantly better than that of E. coli or silkworm baculovirus expression system. Although patent CN113337472A improves the expression level and antiviral activity of FeIFN-omega 2, it still does not solve the problems of short in vivo half-life, lack of long-acting effect and lack of synergistic effect with vaccines of IFN-omega.

[0005] Therefore, there is still an urgent need to develop a new feline IFN-omega preparation technology that has high expression, high stability, long-acting effect and immune-enhancing synergistic effect, in order to meet the clinical needs of feline antiviral protection and immunomodulation. SUMMARY

[0006] The present application aims at solving the problems of low expression efficiency, unstable activity, short half-life and insufficient synergy with vaccines of the existing cat IFN-omega, and provides a recombinantly expressed cat interferon-omega gene, a preparation method and application thereof, which enhances expression, stability and immune level through multiple long-acting modification, CHO system and vaccine compounding, so as to improve the efficacy of cat vaccines.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A recombinantly expressed cat interferon-omega gene, wherein the nucleotide sequence of the recombinantly expressed cat interferon-omega gene is shown in SEQ ID NO. 1.

[0009] Further, the recombinantly expressed cat interferon-omega gene is obtained by codon optimization of a cat interferon-omega natural gene sequence, wherein the cat interferon-omega natural gene sequence is shown in SEQ ID NO. 2.

[0010] Further, the codon optimization is designed by introducing an Fc fusion fragment, introducing an XTEN or PAS extension peptide segment and retaining the original glycosylation modification site on the natural sequence at the C-terminal and / or N-terminal of the cat interferon-omega natural gene sequence.

[0011] Further, the Fc fusion fragment is an Fc region of IgG1 subtype, and the extension peptide segment is a PAS peptide segment.

[0012] A preparation method of a recombinantly expressed cat interferon-omega gene, wherein the method comprises the following steps:

[0013] S1, codon optimizing a cat interferon-omega natural gene sequence to obtain a designed cat interferon-omega gene;

[0014] S2, cloning the cat interferon-omega gene designed in step S1 into an expression vector containing a double promoter and a signal peptide to obtain a recombinant expression vector;

[0015] S3, transfecting the recombinant expression vector in step S2 into a host cell, performing stable clone screening and establishing a high-expression cell strain;

[0016] S4, inoculating the high-expression cell strain in step S3 into a serum-free suspension culture system and performing fermentation culture in a bioreactor, collecting the supernatant to obtain a secreted expression cat interferon-omega fusion protein;

[0017] S5, purifying the cat interferon-omega fusion protein.

[0018] Further, the double promoter in step S2 is a CMV promoter and an EF1 alpha promoter, and the signal peptide is a cat-derived secretion signal peptide.

[0019] Further, the fermentation culture conditions described in step S4 are: pH 7.2-7.4, dissolved oxygen concentration 40-50%, temperature 36.5-37℃, and stirring speed 110-130 rpm.

[0020] Furthermore, the purification process described in step S5 includes: Protein A affinity chromatography, ion exchange chromatography, and molecular sieve chromatography.

[0021] Application of a feline interferon-ω gene prepared by the above method in the preparation of feline vaccines.

[0022] Furthermore, the feline vaccine is effective against one or more of feline calicivirus, feline herpesvirus, and feline panleukopenia.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) High expression efficiency: By combining the CHO-GS stable expression system with dual promoter vectors and signal peptide optimization, the protein yield is significantly improved, reaching more than 100 mg / L, which is far superior to existing expression systems;

[0025] (2) Outstanding long-lasting effect: The use of multiple strategies, including Fc fusion, PAS peptide introduction and glycosylation site modification, extends the half-life compared to conventional cat IFN-ω, reduces the frequency of administration, and improves the convenience and compliance of use.

[0026] (3) Significantly enhances vaccine efficacy: As an adjuvant, it can significantly increase the antibody titer and challenge protection rate induced by the feline vaccine, and comprehensively enhance the vaccine's immune efficacy;

[0027] (4) Good safety: Based on the CHO cell expression system, the protein purity reaches more than 95% after multiple purification steps, which is suitable for the long-term use needs of pets and has better overall performance than existing similar products. Attached Figure Description

[0028] Figure 1 SDS-PAGE electrophoresis image of protein purification;

[0029] Figure 2 Western blotting image for protein purification;

[0030] Figure 3 This is a pharmacokinetic blood drug concentration detection graph;

[0031] Figure 4 This is a graph showing antibody titer detection. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments can be obtained commercially.

[0033] Unless otherwise specified, all percentages mentioned in this invention refer to percentages by mass.

[0034] Example 1: Gene construction and CHO cell expression of long-acting feline interferon-ω

[0035] (1) Gene modification and vector construction

[0036] The natural feline IFN-ω gene sequence was codon-optimized to preserve its core functional activity. A feline IgG1-Fc fragment (SEQ ID NO. 3) was ligated to the C-terminus, and a PAS peptide (SEQ ID NO. 4) was introduced to the N-terminus to enhance protein water solubility and stability. The original glycosylation modification sites on the natural sequence were retained to improve protein stability and pharmacokinetic properties, thus constructing a recombinant gene sequence. The modified gene sequence is shown in SEQ ID NO. 1.

[0037] The modified gene was cloned into the CHO expression vector pCMV-GS containing the CMV and EF1α dual promoters and a pre-defined cat-derived secretory signal peptide sequence to construct the recombinant vector pCHO-IFNω-Fc-PAS.

[0038] (2) Cell transfection and stable line selection

[0039] Log-growth phase CHO-K1 GS cells (American Center of Type Culture Collection) were used. CCL-61 TM The pCHO-IFNω-Fc-PAS recombinant vector was transfected into cells. Single clones were screened by limiting dilution in CD CHO medium (purchased from Gibco 10743029) containing 50 μM MSX. Cells were diluted to 0.5 cells / well and seeded into 96-well plates. After culturing for 2 weeks, the expression level of IFN-ω in the supernatant was detected by ELISA. The single clone with the highest expression level was selected to establish a cell seed bank.

[0040] (3) Suspension culture and protein expression

[0041] Serum-free suspension culture was performed in a 10L bioreactor under the following conditions: temperature 37℃, pH maintained at 7.3, dissolved oxygen level controlled at 45%, and stirring speed 120 rpm. After 7 days of culture, the supernatant was harvested and the expression level was analyzed. The results showed that the yield of the target protein remained stable at over 5000 mg / L.

[0042] Example 2 Protein purification and activity identification

[0043] (1) Protein purification

[0044] The supernatant obtained in Example 1 was used to purify the antigen protein sequentially using Protein A affinity chromatography, ion exchange chromatography, and other purification processes, ultimately obtaining a protein with a purity >95%.

[0045] The purified protein was subjected to SDS-PAGE electrophoresis, stained with Coomassie Brilliant Blue R-250, and then observed. Figure 1 As shown, the results show a single, clear main band with no significant extraneous protein bands, indicating that the protein is not significantly contaminated by exogenous impurities.

[0046] The above electrophoresis samples were subjected to specific Western blotting detection, such as... Figure 2 As shown, a specific band appeared only at the target molecular weight position, and no degradation fragments or non-target protein bands were observed, confirming that the obtained main band was the complete cat IFN-ω fusion protein.

[0047] (2) In vitro antiviral detection

[0048] CRFK cat kidney cells (American Center for Type Culture Collection) were obtained. CCL-94 TM Cell suspension was seeded into 96-well cell culture plates, with 100 μL of cell suspension added to each well. The plates were then incubated at 37 ± 0.5 °C in a 5% CO2 incubator until a dense monolayer formed. Long-acting feline interferon-ω protein prepared in Example 1 and conventional feline IFN-ω protein (obtained from the natural ω sequence according to the method of this invention) were each prepared into 5 μg / mL working solutions as test sample solutions. The original culture medium in the cell culture plate was discarded, and 100 μL of the test sample solution was added to each well. Each sample was set up in 11 parallel wells, and a cell negative control group and a virus positive control group (containing only CRFK feline kidney cells and VSV virus) were also set up. After incubating the culture plates at 37 ± 0.5 °C in a 5% CO2 incubator for 24 h, 100 TCID50 was added to each well. 50 100 μL of VSV virus solution was added and incubated for 24 h. The cytopathic effect was observed and recorded.

[0049] The results are shown in Table 1. Under the same concentration conditions, the long-acting feline interferon-ω protein showed a significantly higher inhibition rate against VSV virus than the conventional feline IFN-ω protein, with an inhibition rate exceeding 90%, indicating that it has a superior and sustained antiviral effect.

[0050] Table 1 Results of in vitro antiviral detection

[0051] Experimental grouping Concentration (pg / mL) Number of lesioned holes Inhibition rate (%) Long-acting IFN-ω 5 1 90.9 Conventional IFN-ω 5 4 63.6 Cell negative control group / 0 100 Virus positive control group / 11 0

[0052] (3) In vivo pharmacokinetic analysis

[0053] Serum cat IFN-ω concentration was determined using a double-antibody sandwich ELISA method, and a standard curve was established. The linear range covered 0.156–10 ng / mL, and the correlation coefficient R was [value missing]. 2 >0.99. SPF-grade BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), weighing 18-22g, were used in the experiment. They were not treated with any drugs before the experiment and had free access to food and water. The mice were randomly divided into 2 groups of 5 mice each.

[0054] Group A: Injected with the long-acting feline interferon-ω prepared in this invention;

[0055] Group B: Injection of routine feline IFN-ω.

[0056] A working solution of 5 μg / mL was prepared using sterile physiological saline and administered via tail vein injection at a dose of 5 μg / kg. Blood samples of 100 μL were collected via the orbital vein at 10, 30, 60, 120, 240, 480, and 720 min after administration. After coagulation at 4°C, the blood samples were centrifuged at 4700 rpm for 10 min to obtain serum. Serum samples were diluted using PBS buffer (1% BSA and 0.05% Tween-20) at dilution ratios ranging from 1:2 to 1:10. A suitable dilution ratio of 1:5 was determined based on preliminary experiments. The serum drug concentration at each time point was determined using a double-antibody sandwich ELISA method according to a standard curve.

[0057] The results are as follows Figure 3 As shown, the long-acting cat IFN-ω prepared by this invention has a significantly longer half-life in vivo than conventional cat IFN-ω, reaching more than 3 times that of conventional cat IFN-ω, indicating that it has a longer retention time and a slower clearance characteristic in vivo.

[0058] Example 3: Validation of the efficacy of long-acting feline IFN-ω as a vaccine adjuvant

[0059] (1) Preparation of vaccine adjuvants

[0060] First, purified long-acting IFN-ω (5 μg / dose) was mixed with feline triple subunit antigens expressed by CHO (feline calicivirus FCV, feline herpesvirus FHV-1, and feline panleukopenia virus FPV = 1:1:1). Then, 4.3% squalene, 0.5% Tween 80, 0.5% Span 85, and citrate buffer were thoroughly mixed and emulsified, and processed using a high-pressure homogenizer to form a stable MF59 oil-in-water nanoemulsion system with droplet sizes between 50 and 200 nm. Finally, the antigen-interferon mixture was mixed with the nanoemulsion at a volume ratio of 1:1, homogenized, emulsified, and aseptically dispensed.

[0061] (2) Immunotherapy efficacy detection

[0062] 30 healthy kittens were randomly divided into 3 groups:

[0063] Group A: Injected with the long-acting IFN-ω vaccine prepared according to this invention;

[0064] Group B: Received a vaccine prepared using the above method, but without the addition of long-acting IFN-ω;

[0065] Group C: Injected with physiological saline as a negative control.

[0066] Each experimental cat received an initial 1 mL injection on day 0 and a booster immunization of 1 mL on day 21.

[0067] Blood samples were collected from the experimental cats in each group on days 14 and 35 post-immunization. Serum was separated and treated in a 56°C water bath for 30 minutes. The neutralizing antibody levels of FHV-1, FCV, and FPV in the serum of each group were detected by a virus neutralization test (see the Chinese Pharmacopoeia for the experimental procedure).

[0068] Heat-inactivated serum samples were serially diluted twofold (1:2, 1:4, 1:8...1:2048); the diluted serum samples were then mixed with equal volumes of FCV, FHV-1, and FPV viral solutions (100 TCID50 each). 50 Mix well and incubate at 37°C for 1 hour to allow the antibody to fully bind to the virus; pre-seed CRFK cells in 96-well plates and culture them in MEM medium with 10% FBS until the cells adhere to the plate at approximately 80%; after removing the original culture medium, add the above virus-serum mixture to each well at 100 μL and gently shake well; also set up blank control wells (medium medium + cells); place the plate in a 37°C, 5% CO2 incubator and incubate for 3-7 days, observing the cytopathic effect daily.

[0069] The results are as follows Figure 4 As shown, Figure 4The geometric mean titers of neutralizing antibodies in each experimental group at different time points after immunization were shown. On day 14 and day 35 after immunization, the neutralizing antibody titers of group A against the three viruses were significantly higher than those of groups B and C, and the antibody response continued to rise. This indicates that the IFN-ω prepared in this invention can significantly enhance the immunogenicity of the vaccine and induce higher levels and longer-lasting neutralizing antibodies.

[0070] (3) Virus attack protection

[0071] 30 healthy kittens were randomly divided into 3 groups:

[0072] Group A: Injected with the long-acting IFN-ω vaccine prepared according to this invention;

[0073] Group B: Received a vaccine prepared using the above method, but without the addition of long-acting IFN-ω;

[0074] Group C: Injected with physiological saline as a negative control.

[0075] Each experimental cat received an initial 1 mL injection on day 0 and a booster immunization of 1 mL on day 21.

[0076] On day 35 post-immunization, experimental cats in each group were simultaneously challenged with a mixture of 1 mL (TCID1 for each virus) of the corresponding virulent strains (FCV, FHV-1, FPV). 50 ≥10 6.0 ), and observe for 14 consecutive days.

[0077] Protection rate = (Number of cats in this group that did not develop the disease after challenge / Total number of experimental cats in this group) × 100%

[0078] The results are shown in Table 2. The results show that the protection rate of group A reached 100%, which is 30% higher than that of group B, indicating that the IFN-ω prepared in this invention can significantly enhance the protective efficacy of the vaccine against mixed infections of multiple viruses.

[0079] Table 2 Results of the challenge protection test

[0080] Group Total number of experimental cats (n) Number of non-diseased cats (n) Protection rate (%) Group A 10 10 100 Group B 10 7 70 Group C 10 0 0

[0081] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A recombinantly expressed feline interferon-ω gene, characterized in that, The nucleotide sequence of the recombinantly expressed feline interferon-ω gene is shown in SEQ ID NO.

1.

2. The recombinant expressed feline interferon-ω gene according to claim 1, characterized in that, The recombinant expressed feline interferon-ω gene was obtained by codon optimization of the natural feline interferon-ω gene sequence; the natural feline interferon-ω gene sequence is shown in SEQ ID NO.

2.

3. The recombinantly expressed feline interferon-ω gene according to claim 2, characterized in that, The codon optimization was achieved by introducing an Fc fusion fragment into the C-terminus and / or N-terminus of the feline interferon-ω natural gene sequence, introducing an XTEN or PAS extended peptide, and retaining the original glycosylation modification sites on the natural sequence.

4. The recombinantly expressed feline interferon-ω gene according to claim 3, characterized in that, The Fc fusion fragment is the Fc region of the IgG1 subtype, and the extended peptide is the PAS peptide.

5. A method for preparing recombinantly expressed feline interferon-ω gene according to any one of claims 1-4, characterized in that, The method includes the following steps: S1. Codon optimization was performed on the natural feline interferon-ω gene sequence to obtain the designed feline interferon-ω gene; S2. The feline interferon-ω gene designed in step S1 is cloned into an expression vector containing a dual promoter and a signal peptide to obtain a recombinant expression vector. S3. Transfect the recombinant expression vector described in step S2 into host cells, perform stable clone screening, and establish a high-expression cell line. S4. The high-expression cell line described in step S3 is seeded into a serum-free suspension culture system, fermented in a bioreactor, and the supernatant is collected to obtain the secreted and expressed cat interferon-ω fusion protein. S5. The cat interferon-ω fusion protein is purified.

6. The preparation method according to claim 5, characterized in that, The dual promoters mentioned in step S2 are the CMV promoter and the EF1α promoter, and the signal peptide is a cat-derived secretory signal peptide.

7. The preparation method according to claim 5, characterized in that, The fermentation conditions described in step S4 are: pH 7.2-7.4, dissolved oxygen concentration 40-50%, temperature 36.5-37℃, and stirring speed 110-130 rpm.

8. The preparation method according to claim 5, characterized in that, The purification process described in step S5 includes: Protein A affinity chromatography, ion exchange chromatography, and molecular sieve chromatography.

9. The use of the feline interferon-ω gene prepared by the method according to any one of claims 5 to 8 in the preparation of feline vaccines.

10. The application according to claim 9, characterized in that, The feline vaccine is effective against one or more of feline calicivirus, feline herpesvirus, and feline panleukopenia.

Images