Recombinant porcine alpha and gamma interferon
Through the insect-baculovirus expression system and synonymous preference modification codons, combined with the use of phomin signal peptides, the defects of the pig α and γ interferon expression systems in the prior art were solved, efficient and stable co-expression and purification were achieved, and biological activity and production efficiency were improved.
Patent Information
- Application Number
- CN202510092737.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-03-28
AI Technical Summary
The existing expression systems have problems such as protein modification, inclusion body existence, and hybrid protein production when expressing pig α and γ interferon, which leads to increased purification difficulty and cost and affects activity.
The insect-baculovirus expression system was adopted to modify the codons through synonymous preferences, add phomin signal peptides to achieve extracellular expression, and recombinant vector construction and viral infection were carried out using pFastBacTMDual vector and DH10Bac cells to achieve efficient co-expression of pig α and gamma interferon.
The efficient expression of pig α and γ interferon is achieved, the protein structure is close to the natural state, the biological activity is high, the purification is easy to carry out, which reduces production costs and improves the quality and stability of the recombinant protein.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of veterinary medicine, and in particular relates to the expression of pig alpha and gamma interferon recombinant proteins. Background Art
[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.
[0003] Existing expression systems such as Escherichia coli or Pichia pastoris have different disadvantages, such as the lack of post-expression modification of the expressed protein, the protein exists in the form of inclusion bodies, which are difficult to purify subsequently, and most of the expressed proteins are accompanied by the production of other impurities, which increases the difficulty and cost of purification, and at the same time damages the activity of interferon, thereby affecting its ultimate application effect.
[0004] The insect-baculovirus expression system is a commonly used eukaryotic expression system in recent years, which has the following advantages: ① The insect baculovirus expression system provides a good environment for the correct folding of exogenous target proteins, the formation of disulfide bonds and oligomers, so that the expressed target proteins have complete biological functions; ② The target proteins can be post-translationally modified, such as phosphorylation, glycosylation, signal peptide removal, etc., to restore the natural state of the target proteins to the greatest extent; ③ The expression level is high, and the expression amount of the target protein can account for up to 30% of the total cell protein; ④ It is highly safe and harmless to humans and animals; ⑤ It can accommodate large molecular weight inserts or express multiple exogenous genes at the same time. Summary of the invention
[0005] In view of the problems in the prior art, the present invention provides a sequence of porcine interferon α and γ recombinant proteins, in which the codons are modified with synonymous preference, and can be applied to insect baculovirus system to express proteins. In addition, a bee pollen signal peptide is added to achieve extracellular expression.
[0006] To achieve the above purpose, the present invention adopts the following technical solution.
[0007] A gene encoding porcine interferon-alpha, the nucleotide sequence of which is shown in SEQ ID NO: 1 or 2.
[0008] A gene encoding porcine interferon-γ, the nucleotide sequence of which is shown in SEQ ID NO: 3 or 4.
[0009] A recombinant vector and cell comprising the above-mentioned coding gene of porcine interferon-α and / or coding gene of porcine interferon-γ. The above-mentioned recombinant vector and cell can express porcine interferon-α and / or porcine interferon-γ; the expression system is preferably an insect baculovirus expression system. The backbone of the recombinant vector is preferably pFastBac TMDual vector; the cells are preferably DH10Bac cells, Sf 9 cells or Sf 21 cells.
[0010] A method for co-expressing porcine interferon-α and interferon-γ comprises the following steps: (1) Transfer the nucleic acids containing the above-mentioned coding genes into pFastBac TM Dual vector, construct recombinant vector; (2) Transform the recombinant vector into DH10Bac competent cell to screen positive clones and extract positive shuttle plasmids; (3) Infect sf9 cells with the shuttle plasmid to obtain recombinant virus; (4) The recombinant virus was used to infect sf9 cells, and porcine interferon α and γ were separated and purified from the culture medium.
[0011] The present invention has the following advantages: The expression system provided by the present invention can simultaneously and efficiently express type I interferon α and type II interferon γ, which not only exerts the antiviral, anti-cell division and immunomodulatory effects of interferon, but also activates T cells and natural killer cells, giving full play to the multiple activities of interferon. The present invention co-expresses two interferons in a eukaryotic expression system, which can fold and process the expressed protein to make the structure of the expressed protein closer to the natural structure and have high biological activity; the recombinant protein is expressed by different promoters, and the expression amount is high and stable; the protein is secreted into the fermentation supernatant under the guidance of the bee pollen signal peptide, effectively avoiding the metabolic load on the host cell caused by excessive expression products. At the same time, the secretory protein produced by insect cells themselves is less, which is easy to purify the recombinant protein. Insect cells can grow in serum-free culture medium, which is easy to expand the production scale, and there is no need to handle live viruses or potentially dangerous pathogens, thereby reducing production costs; by optimizing the production process and fermentation conditions, the quality and stability of the recombinant protein can be further improved. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 It is a schematic diagram of the structure of the recombinant plasmid pFD-α-γ; Figure 2 The morphology of Sf 9 cells not transfected with pBac-α-γ (A) and Sf 9 cells transfected with pBac-α-γ (B); Figure 3 Immunofluorescence images of Sf 9 cells infected with baculovirus (A) and Sf 9 cells not infected with baculovirus (B); Figure 4These are SDS-PAGE (A) and Western-Blot (B) of different cell culture supernatants; where M in A stands for Maker (10-180KDa), 1 is the supernatant of P1 generation, 2 is the supernatant of P2 generation, 3 is the supernatant of P3 generation, and 4 is the supernatant of uninoculated cells; M in B stands for Maker (10-180KDa), 1 is the supernatant of uninoculated cells, 2 is the WB result after the supernatant of P3 generation was combined with anti-α monoclonal antibody, and 3 is the WB result after the supernatant of P3 generation was combined with anti-γ monoclonal antibody. DETAILED DESCRIPTION
[0013] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.
[0014] Example 1 Optimization of the gene encoding porcine interferon The porcine interferon-α gene (NP_999558.1) published on NCBI was modified according to the codon preference of silkworm to obtain the coding gene shown in SEQ ID NO: 1; after adding a peakin signal peptide at the front end and 6 His tags at the end, BamHI and HindIII restriction sites were added at both ends for full synthesis to obtain the interferon-α target gene shown in SEQ ID NO: 2.
[0015] The porcine interferon-γ gene (NP_999113.1) published on NCBI was modified according to the codon preference of silkworm to obtain the coding gene shown in SEQ ID NO: 3; after adding the peakin signal peptide at the front end and 6 His tags at the end, XhoI and KpnI restriction sites were added at both ends for full synthesis to obtain the interferon-γ target gene shown in SEQ ID NO: 4.
[0016] Example 2 Construction of a recombinant vector co-expressing porcine interferon α and γ 1. Recombinant plasmid The interferon-α target gene in Example 1 was ligated to pFastBac TM After the pH promoter in the dual vector, transfect DH5α competent cells, select single colonies after screening and inoculate them into LB liquid medium with ampicillin resistance, place them in a 37℃ shaker overnight, centrifuge the bacterial liquid and extract the plasmid with a plasmid extraction kit. The extracted plasmid is double-digested with restriction endonucleases and PCR is performed to identify the product. The positive plasmid is named pFD-α; In the same way, the interferon-γ target gene in Example 1 was connected to the p10 promoter in the pFD-α vector, and the positive plasmid was named pFD-α-γ. Figure 1 shown.
[0017] 2. Recombinant bacmid The recombinant plasmid pFD-α-γ was transformed into DH10Bac competent medium, and then cultured in a liquid medium without resistance at 37°C for 4 hours. Then, a bacterial suspension was prepared and spread on LB blue-white triple-antibody screening solid medium, and inverted at 37°C for 48 hours. White-spotted colonies were picked and streaked on blue-white LB solid medium for screening for purification and screening. After repeated screening, white plaques were picked and placed in liquid medium with the same resistance, shaken and cultured at 37°C for 16 hours, and the recombinant bacmid in the bacterial solution was extracted. PCR and double enzyme digestion were used to identify the size of the target fragment and sequenced. The positive plasmid was named pBac-α-γ.
[0018] Example 3 Co-expression of porcine interferon α and γ 1. Obtaining Baculovirus The recombinant bacmid pBac-α-γ in Example 2 was transfected into Sf 9 cells in the logarithmic growth phase and cultured at 27°C for 96-120 hours. Compared with the normal adherent Sf 9, it was observed under a microscope that the transfected Sf 9 cells were enlarged and rounded, the nucleus filled the entire cell, and the cells dissociated in the late culture period, etc. ( Figure 2 ), harvest the supernatant of the diseased Sf9 cells, which is the P1 generation baculovirus.
[0019] 2. Baculovirus Titer Sf9 cells were cultured at 27°C for 12 h and then replaced with serum-free medium. P1 virus solution was inoculated at 1% volume per bottle and cultured in a 27°C incubator for 72 h. The cell culture supernatant was collected and centrifuged at 4°C and 3000 rpm for 30 min to remove cell debris and obtain P2 virus solution. The recombinant baculovirus was blindly propagated to P4 according to this method, and the genomic DNA of P4 cells was extracted and used as a template for PCR identification. TCID 50 Methods The titer of recombinant baculovirus was determined and calculated by the Reed-Muench method.
[0020] Table 1 P4 virus titer determination According to the data in Table 1, the virus titer of P4 generation is 1×10 6.49 IFU / mL.
[0021] 3. Protein Expression Analysis The P4 generation was inoculated into adherent Sf 9 cells and cultured for 48 h before indirect immunofluorescence analysis. Figure 3 As shown: Obvious green fluorescence can be observed under a fluorescence microscope after baculovirus inoculation, while no fluorescence appears in uninfected cells; this indicates that Sf 9 cells infected with baculovirus can co-express α and γ interferons and specifically react with anti-α and γ monoclonals, respectively.
[0022] The supernatants of normal Sf 9 cells and baculovirus-infected Sf 9 cells were subjected to SDS-PAGE electrophoresis and Western-Blot analysis. Figure 4 As shown. After inoculation of P1, P2, and P3 viruses, the target protein was produced, with a size of about 20 kDa, which is consistent with the expected size ( Figure 4 A); The target protein can be bound to anti-α and γ monoclonal antibodies ( Figure 4 B); This indicates that the constructed baculovirus-insect cell expression system can co-express α and γ interferons.
[0023] 4. Antiviral potency of porcine interferon-α and interferon-γ PK-15 cells (pig kidney cells), BHK-21 cells (hamster kidney cells), ST cells (pig testicular cells), MRAC145 cells (African green monkey embryonic kidney cells), and Vero cells (African green monkey kidney cells) were used as test cells to determine the titer of co-expressed porcine interferon α and γ against porcine circovirus 2d subtype (PCV2d), porcine Seneca virus (SVA), porcine epidemic diarrhea virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine transmissible gastroenteritis virus (PEDV), and pseudorabies virus (PRV); the titer was determined by the cytopathic effect inhibition method, and the titer unit was calculated based on 50% lesion. The specific method is as follows: Interferon purification: Due to the action of the beekeeper signal peptide, the cell interferon secreted and expressed is collected from the supernatant, centrifuged at 3000rmp / min to remove cell debris, and the pH value is adjusted to 7.2 after condition exploration to promote protein precipitation during subsequent purification. Interferon is purified using an affinity chromatography column with a His tag.
[0024] Discard the culture medium from the wells with cells that have grown into a good monolayer, use cell growth medium to make a 10-fold serial dilution of the purified interferon sample, inoculate 8 wells for each dilution, 0.1 mL per well, place in an incubator at 37°C with 5% CO2, culture for about 20 hours, pour off the sample, wash twice with maintenance solution, and then inoculate the virus; set up an interferon control group, a cell control group, and a virus control group.
[0025] The virus solution was diluted to 1000 TCID 50 / mL and 100TCID 50 / mL, add 0.1mL to each well of the culture plate, and culture in a 37℃, 5% CO2 incubator for 72h.
[0026] According to the Reed-Muench method, interferon units are log 10The reciprocal of the highest dilution of interferon that can protect half of the cells from being destroyed by the attacking virus is the interferon titer.
[0027] Table 2 The potency of porcine interferon α and γ against different viruses From the above data, it can be seen that the separated mixed samples of porcine interferon α and γ have good activity against 7 types of porcine viruses.
[0028] Application Example 1: Treatment of piglet diseases caused by circovirus with porcine interferon α and γ 1. Experimental Methods The recombinant interferon purified from the cell supernatant was determined to have a concentration of (10 7 U / mL).
[0029] The healthy susceptible piglets with negative PCV2 antigen and negative PCV2 antibody at 14-21 days of age were randomly divided into 7 groups, with groups 1-4 as the challenge treatment group, group 5 as the emergency prevention challenge group, group 6 as the challenge non-treatment group, and group 7 as the blank control group. There were 5 pigs in each group, and a total of 35 pigs were used in the experiment. The piglets were randomly divided into groups and isolated for breeding, and the experiment began on the second day.
[0030] On the first day of the experiment, all experimental pigs in groups 1-5 were injected intramuscularly with 5.0 mL of keyhole limpet hemocyanin emulsified with Freund's incomplete adjuvant (KLH / ICFA, 0.5 mg / mL) and intraperitoneally with 10 mL of thioglycollate medium. The emergency prevention and challenge group in group 5 was injected with 4 mL of recombinant porcine α-interferon every day, injected intramuscularly in the neck for three consecutive days.
[0031] On the fourth day of the experiment (the first day of the challenge), pigs in groups 1-6 were inoculated with PCV2 (SD strain) (containing 10 6.0 TCID 50 / mL) virus culture fluid, the challenge dose was 2.0mL per pig with intranasal drops and 3.0mL for intramuscular injection. On the 3rd and 6th days after the challenge, all test pigs in groups 1-6 were injected intramuscularly with 5.0mL / head of keyhole limpet hemocyanin emulsified with Freund's incomplete adjuvant (KLH / ICFA, 0.5mg / mL), and 10mL / head of thioglycolic acid culture medium was injected intraperitoneally. All piglets were weighed on the day of infection and on the 28th day after infection. The body temperature of piglets was measured daily for the first 7 days after infection. The test was terminated on the 28th day after infection. Blood from the anterior vena cava of all pigs was collected for PCR testing, and the disease was determined based on the body temperature, relative daily weight gain and virus antigen results.
[0032] After the onset of symptoms, Group 1 received 2 mL of recombinant porcine interferon (10 7 U / mL) was injected into the neck muscle for three consecutive days; the second group was injected with 4 mL of recombinant porcine interferon (10 7U / mL) was injected into the neck muscle for three consecutive days; the third group was injected with 6mL recombinant porcine interferon (10 7 U / mL) was used for treatment, injected intramuscularly in the neck for three consecutive days; the fourth group was the positive control substance - porcine leukocyte interferon (2×10 4 U / mL) treatment, injected into the neck muscle for three consecutive days; the treatment effect was evaluated after 28 days of observation. Groups 5 and 6 did not receive other treatments, and the effect after the challenge was directly evaluated.
[0033] Table 3 Piglet groups and treatments 2. Effect evaluation The therapeutic effect and emergency prevention effect were evaluated separately according to the effect evaluation criteria.
[0034] The piglets in groups 1-4 and groups 6 and 8 were weighed on the first day and 28 days after treatment. The body temperature of the piglets was measured daily during the first 7 days after treatment. After the treatment was terminated on the 28th day, all pigs were subjected to PCR testing of inguinal lymph nodes. The results of body temperature, relative daily weight gain and viral antigen were used to determine whether the piglets were protected by treatment. If the following three items were met, the piglets were considered protected by treatment: (1) Body temperature standard: elevated body temperature (≥40°C) (piglets’ body temperature is measured once a day at around 10 a.m.) and must not persist for more than 3 days (not including 3 days); (2) Relative daily weight gain standard: On the first day and the 28th day after treatment, the test pigs in Groups 1-4 and Group 6 were weighed respectively, and the relative daily weight gain of the five groups of test pigs was calculated within 28 days. If the relative daily weight gain of the test pigs in the non-treatment group was significantly lower than that in the treatment group (P < 0.05), all pigs in this group were determined to be protected by treatment.
[0035] Relative daily weight gain = (body weight 28 days after challenge - body weight on the day of challenge) / 28 / body weight on the day of challenge.
[0036] P value was calculated and t test of two independent samples was performed using biostatistical software.
[0037] On the first day and the 28th day after treatment, the weights of the experimental pigs in Groups 1-4 and Group 7 were weighed respectively, and the relative daily weight gain of the experimental pigs in the five groups was calculated within 28 days. If the relative daily weight gain of the experimental pigs in Group 4 was not significantly different from that in Group 7 (P>0.05), all the pigs in the group were determined to be protected by treatment.
[0038] Relative daily weight gain = (body weight 28 days after challenge - body weight on the day of challenge) / 28 / body weight on the day of challenge.
[0039] P value was calculated and t test of two independent samples was performed using biostatistical software.
[0040] (3) Virus antigen detection: No PCV2 antigen should be detected when testing the inguinal lymph node tissue of piglets using the PCR method.
[0041] Group 5 will be judged 28 days after the start of the experiment. If the following three items are met, it can be judged as emergency preventive protection: (1) Body temperature standard: The body temperature should be elevated (≥40℃) (measure the piglet’s body temperature once a day at around 10 am) and should not last for more than 3 days (not including 3 days).
[0042] (2) Relative daily weight gain standard: Weigh the pigs in Group 5 and Group 7 on the day of infection and 28 days after infection, and calculate the relative daily weight gain of the two groups of pigs within 28 days after infection. If the relative daily weight gain of the pigs in Group 5 is not significantly different from that in Group 7 (P>0.05), all pigs in this group are considered to be protected from treatment.
[0043] Relative daily weight gain = (body weight 28 days after challenge - body weight on the day of challenge) / 28 / body weight on the day of challenge.
[0044] P value was calculated and t test of two independent samples was performed using biostatistical software.
[0045] (3) Virus antigen detection: No PCV2 antigen was detected in piglet blood using the PCR method.
[0046] Table 4 Body temperature changes of experimental pigs after virus attack As can be seen from Table 4, the porcine circovirus type 2 SD strain virus solution (10 5.5 TCID 50 / mL) of the test pigs, the body temperatures of 2 test pigs in Group 1 increased (≥40℃) and lasted for 3 days; the body temperatures of 3 test pigs in Group 2 increased (≥40℃) and lasted for 3 days; the body temperatures of 2 test pigs in Group 3 increased (≥40℃) and lasted for 3 days; the body temperatures of 4 test pigs in Group 4 increased (≥40℃) and lasted for 3 days; the body temperatures of 4 test pigs in Group 6 increased (≥40℃) and lasted for 3 days; the body temperatures of the test pigs in Groups 5 and 7 did not change significantly.
[0047] Table 5 Weight changes of experimental pigs after virus challenge As can be seen from Table 5, the pigs in Groups 1-4 and 6 were treated with porcine circovirus type 2 SD strain virus liquid (10 5.5 TCID 50 / mL) after virus attack, the statistical software SPSS20.0 analyzed that the relative daily weight gain of group 1 was compared with that of the blank control group, P<0.05, and the difference was significant. All pigs in this group had clinical symptoms caused by circovirus type 2; the relative daily weight gain of group 2 was compared with that of the blank control group, P<0.05, and the difference was significant. All pigs in this group had clinical symptoms caused by circovirus type 2; the relative daily weight gain of group 3 was compared with that of the blank control group, P<0.05, and the difference was significant. All pigs in this group had clinical symptoms caused by circovirus type 2. clinical symptoms caused by type 2; the relative daily weight gain of group 4 was compared with that of the blank control group, P<0.05, with significant difference, and all pigs in this group had clinical symptoms caused by circovirus type 2; the relative daily weight gain of group 6 was compared with that of the blank control group, P<0.05, with significant difference, and all pigs in this group had clinical symptoms caused by circovirus type 2; the relative daily weight gain of group 5 treated and attacked groups was compared with that of the blank control group, P>0.05, with no significant difference, and all pigs in this group had no clinical symptoms caused by circovirus type 2.
[0048] Table 6 Antigen detection table of experimental pigs after virus challenge As can be seen from Table 6, PCR testing of the experimental pigs 28 days after infection showed that 3 pigs in Group 1, 4 pigs in Group 2, 3 pigs in Group 3, 4 pigs in Group 4, 1 pig in Group 5, and 4 pigs in Group 6 were antigen-positive, while all pigs in Group 7 were antigen-negative.
[0049] Table 7 Results of interferon challenge test Note: “+” indicates positive; “-” indicates negative; “\” indicates negative.
[0050] The test results showed that the incidence rates of group 1, group 2, group 3, group 4 and group 6 were 5 / 5, 5 / 5, 4 / 5, 5 / 5 and 5 / 5 respectively, and the incidence rate of group 5 was 0 / 5, which proved that the attack model was successful, the treatment effect of group 5 was better, and no disease occurred after the attack.
[0051] Table 8 Body temperature changes of experimental pigs after treatment As can be seen from Table 8, the experimental pigs were treated with porcine circovirus type 2 SD strain virus solution (10 5.5 TCID 50 / mL) after the onset of the disease, interferon was used for three consecutive days. The body temperature of the test pigs in Groups 1-4 did not change significantly (≤40℃); the body temperature of two test pigs in Group 6 increased (≥40℃), but it did not last for 3 days.
[0052] Table 9 Weight changes of experimental pigs after treatment As can be seen from Table 9, after the 1st to 4th groups of experimental pigs were treated with interferon, the statistical software SPSS20.0 was used to analyze the relative daily weight gain of group 1 compared with the relative daily weight gain of the non-treatment group, P<0.05, and the difference was significant. The effect of interferon treatment on all pigs in this group was significantly improved; the relative daily weight gain of group 2 was compared with the relative daily weight gain of the non-treatment group, P<0.05, and the difference was significant. The effect of interferon treatment on all pigs in this group was significantly improved; the relative daily weight gain of group 3 was compared with the relative daily weight gain of the non-treatment group, P<0.05, and the difference was significant. The effect of interferon treatment on all pigs in this group was significantly improved; the relative daily weight gain of group 4 was compared with the relative daily weight gain of the non-treatment group, P<0.05, and the difference was significant. The effect of interferon treatment on all pigs in this group was significantly improved.
[0053] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims
1. A gene encoding porcine interferon-α, characterized in that: The nucleotide sequence is shown in SEQ ID NO: 1 or 2.
2. A gene encoding porcine interferon-γ, characterized in that: The nucleotide sequence is shown in SEQ ID NO: 3 or 4.
3. A recombinant vector and cell comprising the above-mentioned gene encoding porcine interferon-α and / or gene encoding porcine interferon-γ.
4. The recombinant vector and cell according to claim 3, characterized in that: Co-expression of porcine interferon-α and porcine interferon-γ.
5. The recombinant vector and cell according to claim 4, characterized in that: The expression system is an insect baculovirus expression system; the backbone of the recombinant vector is pFastBac TM Dual vector; the cells are selected from DH10Bac cells, Sf 9 cells or Sf 21 cells.
6. A method for co-expressing porcine interferon α and γ, characterized in that: The following steps are involved: (1) The nucleic acids containing the coding genes of claims 2 and 4 were respectively transferred into pFastBac TM Dual vector, construct recombinant vector; (2) Transform the recombinant vector into DH10Bac competent cell to screen positive clones and extract positive shuttle plasmids; (3) Infect sf9 cells with the shuttle plasmid to obtain recombinant virus; (4) The recombinant virus was used to infect sf9 cells, and porcine interferon α and γ were separated and purified from the culture medium.
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