Recombinant pigeon interferon alpha gene, recombinant expression vector thereof, recombinant host bacteria and preparation method of recombinant pigeon alpha interferon

Abstract

The application belongs to the field of genetic recombination and veterinary medicine, and discloses a recombinant pigeon interferon alpha gene, a recombinant expression vector of the pigeon interferon alpha gene, a recombinant host bacterium and a preparation method of the recombinant pigeon interferon alpha, wherein the nucleotide sequence of the recombinant pigeon interferon alpha gene is shown as SEQ ID NO. 1. The recombinant expression vector is recombined by an expression vector and the recombinant pigeon interferon alpha gene. The recombinant host bacterium is transformed with the recombinant expression vector, and the preparation method of the recombinant pigeon interferon alpha comprises the following steps: fermentation culture is carried out on the recombinant host bacterium, the fermentation liquid supernatant is collected, and the recombinant pigeon interferon alpha is obtained through purification. The preparation method of the recombinant pigeon interferon alpha provided by the application is not only stable in process, but also greatly improves the expression amount and the antiviral activity of the pigeon interferon alpha compared with the shake flask culture mode, and is suitable for large-scale production application. In addition, the method also reduces the production cost of the pigeon interferon alpha, and the Pichia pastoris host bacterium used in the method has a long survival time under high-density growth conditions, and is suitable for large-scale production application.

Description

Technical Field

[0001] This invention relates to the fields of gene recombination and veterinary drugs, and particularly to a recombinant pigeon interferon α gene, its recombinant expression vector, recombinant host bacteria, and a method for preparing recombinant pigeon α interferon. Background Technology

[0002] Pigeon alpha interferon, a cytokine with broad-spectrum antiviral and immunomodulatory activities, has enormous application potential in the prevention and treatment of pigeon diseases. Currently, the production of recombinant pigeon alpha interferon through genetic engineering has become a research hotspot.

[0003] The Pichia pastoris expression system has many advantages, such as: it has a strong methanol-inducible AOX (alcohol oxidase gene) promoter, which can strictly regulate the expression of exogenous genes; the culture cost of this system is low, the fermentation medium used is mainly composed of glycerol, methanol and inorganic salts, and the products are easy to separate; the exogenous protein gene in the Pichia pastoris expression system has high genetic stability and can be integrated into the Pichia pastoris genome at a high copy number, thereby obtaining high-expression strains; at the same time, because Pichia pastoris has a subcellular structure of eukaryotes, it can perform post-translational modifications such as glycosylation on the expressed protein, thereby improving protein stability and activity, making it an ideal host system for expressing recombinant pigeon α-interferon.

[0004] However, existing production processes using Pichia pastoris to express recombinant pigeon α-interferon still have some problems. For example, the expression level of this recombinant protein is low, making it difficult to meet the needs of large-scale industrial production; the utilization efficiency of nutrients such as carbon and nitrogen sources during fermentation is low, leading to increased fermentation costs; the fermentation cycle is long, affecting production efficiency; in addition, during fermentation, the lack of precise control over environmental factors (such as temperature, pH, dissolved oxygen, etc.) and induction conditions (such as the timing, amount, and frequency of methanol addition) results in unstable biological activity of recombinant pigeon α-interferon and significant batch-to-batch variations. Currently, the highest potency of pigeon α-interferon expressed in publicly available data (patent application by Jiangsu Academy of Agricultural Sciences with authorization announcement number CN116143900B) is 3.16 × 10⁻⁶. 6 IU / ml.

[0005] Therefore, developing a high-efficiency, stable, and low-cost fermentation process for expressing recombinant pigeon α-interferon in Pichia pastoris is of great practical significance. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a recombinant pigeon interferon α gene, its recombinant expression vector, a recombinant host bacterium, and a method for preparing recombinant pigeon α interferon. The recombinant pigeon interferon α gene can be efficiently expressed in a yeast expression system, and the method for preparing recombinant pigeon interferon α provides a large-scale fermentation process that can significantly increase yield and improve the potency of recombinant pigeon α interferon in the fermentation broth. This method is suitable for large-scale production and provides a solution for the industrial production of recombinant pigeon α interferon.

[0007] To address the aforementioned problems in the prior art, the technical solution provided by this invention is as follows: In a first aspect, this application provides a recombinant pigeon interferon α gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] To adapt to the characteristics of the yeast expression system, the recombinant pigeon interferon α gene was obtained by removing the signal peptide from the pigeon interferon α gene (GenBank accession number: MG_833835.1) and optimizing the sequence to suit the codon preferences of Pichia pastoris. This recombinant pigeon interferon α gene can be expressed normally and efficiently in Pichia pastoris.

[0009] Secondly, this application also provides a recombinant expression vector carrying the pigeon interferon α gene, which is formed by recombining the expression vector with the aforementioned recombinant pigeon interferon α gene.

[0010] Optionally, the expression vector is a pPIC9K series plasmid, such as pPIC3.5K, pPIC9K, etc.

[0011] The method for constructing the recombinant expression vector is as follows: appropriate restriction sites (such as EcoRI and NotI) are added to both ends of the aforementioned recombinant pigeon interferon α gene, and the sequence is then constructed into the pPIC9K vector. The recombinant expression plasmid pPICPiIFN-α is prepared by restriction enzyme ligation / direct synthesis.

[0012] Thirdly, this application also provides a recombinant host bacterium, in which the aforementioned recombinant expression vector is transformed.

[0013] Optionally, the recombinant host strain is Pichia pastoris, such as Pichia pastoris GS115.

[0014] Pichia pastoris expression system is one of the most reliable exogenous gene expression systems currently available, and it has replaced traditional E. coli and Saccharomyces cerevisiae expression systems in the industrial production of many exogenous proteins. First, the Pichia pastoris expression system contains a strong AOX (alcohol oxidase gene) promoter, allowing for precise regulation of exogenous gene expression using methanol. Second, it has low cultivation costs and allows for soluble product expression. The fermentation medium used for Pichia pastoris is cost-effective, typically containing glycerol or glucose and methanol as carbon sources, with the remainder being inorganic salts. The medium is protein-free, which facilitates downstream product separation and purification. Third, the exogenous protein gene is genetically stable, integrating into the Pichia pastoris genome at a high copy number, minimizing loss and yielding highly expressed strains. Therefore, Pichia pastoris is the preferred expression system for recombinant pigeon α-interferon bacteria, not only reducing production costs and purification difficulty but also significantly improving the potency of the active ingredient.

[0015] Fourthly, this application also provides a method for preparing recombinant pigeon α-interferon, which includes the following steps: The aforementioned recombinant host bacteria were fermented and cultured, and the supernatant of the fermentation broth was collected and purified to obtain recombinant pigeon α-interferon.

[0016] Optionally, in the preparation method of the recombinant pigeon α-interferon, the fermentation culture method includes the following steps: (1) Growth stage: Add 1~2.5L of fermentation medium to the fermentation equipment, and then inoculate the seed liquid of the above-mentioned recombinant host bacteria into the fermentation system. The aeration rate is 3-6L / min, the rotation speed is 250-650 rpm, the temperature is 28-30℃, micro-oxygenation is used to maintain the dissolved oxygen content of DO at 20%-50%, and the pH is maintained at 6.0±0.05. The culture time of this stage is 20~23h.

[0017] (2) Glycerol feeding stage: When the culture is about 24h-28h, add 50% glycerol containing 1-8‰ PTM1 by mass volume at a rate of 13-18mL / h / L, continue to culture for 3-5h, stop feeding, and when the glycerol in the tank is exhausted, starve culture for 45-90min; (3) Induction expression stage: The pH was adjusted to 6.0±0.05, and methanol was added in stages at a flow rate of 5 mL / h / L~10 mL / h / L to induce fermentation. The dissolved oxygen content was maintained at 12%-55% by dynamically adjusting the rotation speed and aeration rate until the fermentation ended after 78 hours. 1-8‰ PTM1 was added to the methanol to supplement trace elements.

[0018] Preferably, the inoculation amount of the host bacteria seed liquid is 5% (volume ratio, seed liquid OD600 is 4-8).

[0019] Preferably, the volume of the fermentation medium is 2.2L, and the rotation speed is 250-650 rpm, with a preferred rotation speed of 550 rpm.

[0020] Optionally, the fermentation medium is BSM medium.

[0021] Preferably, during the growth stage, the temperature is set to 28-30℃, more preferably 30℃; during the induction expression stage, the temperature is set to 28±0.5℃.

[0022] Optionally, in the method for preparing recombinant pigeon α-interferon, the method for the induction expression stage specifically includes: The pH was adjusted to 6.0±0.05, methanol was added, and induction was started at a flow rate of 5.0±1.0 mL / h / L for the initial fermentation medium. The dissolved oxygen level was maintained at 12%-55% by dynamically adjusting the aeration rate and the rotation speed. After 24 h of induction, methanol was added at a flow rate of 6.0±1.0 mL / h / L. After 48 h of induction, methanol was added at a flow rate of 7.0±1.0 mL / h / L. After 72 h of induction, methanol was added at a flow rate of 8.0±1.0 mL / h / L until fermentation ended at 78 h.

[0023] Optionally, in the method for preparing recombinant pigeon α-interferon, the method for constructing the recombinant host bacterium is as follows: The pigeon interferon α gene with the nucleotide sequence shown in SEQ ID NO.1 was cloned into an expression vector to construct a recombinant expression vector, which was then transformed into a host bacterium to obtain a recombinant host bacterium.

[0024] Fifthly, this application also provides the application of recombinant pigeon α-interferon prepared by the above method in determining its antiviral activity in the chicken embryo fibroblast-vesicular stomatitis virus system.

[0025] The present invention has the following beneficial effects: 1. The fermentation method of this application significantly improves the expression level of recombinant pigeon α-interferon: Through a series of measures such as optimizing the fermentation medium formula, precisely controlling the fermentation process parameters, and adopting a segmented induction strategy, the fermentation method of this invention increases the expression level of recombinant pigeon α-interferon by about 10 times compared with the traditional process. The expression level of recombinant pigeon α-interferon can reach 790~850 mg / L of fermentation broth, which meets the needs of large-scale industrial production.

[0026] 2. The fermentation method of this application enhances the biological activity of recombinant pigeon α-interferon: The optimized fermentation process ensures that the recombinant pigeon α-interferon possesses good biological activity. Its antiviral activity has been measured to reach 3.16 × 10⁻⁶. 8The concentration of IU / mL significantly enhances immunomodulatory activity, reaching 100 times the highest potency of pigeon α-interferon expressed in Pichia pastoris in existing literature, far exceeding current technological levels. It can effectively stimulate the proliferation of peripheral blood lymphocytes in pigeons and enhance their immunity.

[0027] 3. The fermentation method of this application can reduce production costs and shorten the fermentation cycle: This invention improves the utilization efficiency of nutrients and reduces fermentation costs by optimizing the nutrient formula and adopting a staged continuous feeding fermentation strategy. At the same time, precise fermentation process control and optimized induction conditions shorten the fermentation cycle from 7-9 days in traditional processes to 5-6 days, greatly improving production efficiency.

[0028] 4. The fermentation method of this application enhances the stability and repeatability of the fermentation process: This invention precisely controls the key parameters in the fermentation process, reduces batch-to-batch differences, and makes the fermentation process have good stability and repeatability, which is beneficial to the quality control of industrial production. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0030] Figure 1 The linearized plasmid pPIC9K-PiIFN was electrotransformed into Pichia pastoris GS115. After selection for genimycin resistance, a portion of single clones were selected for PCR identification; M is the DL5000 DNA Marker. Figure 2 Western blot identification results of the expression product of recombinant strain GS115 / pPIC9K-PiIFN-α; lanes 1-6 are the culture supernatant of the clone after 96 h of induction, lane 7 is the culture supernatant of Pichia pastoris GS115 / pPIC9K after 96 h of induction, M is the molecular weight marker of the prestained protein; the target band is between 25 KD and 35 KD; Figure 3 : Induction time and yeast cell density changes of recombinant bacteria GS115 / Ppic9K-PiIFN-α in 5L bioreactor and shake flask culture, respectively; Figure 4 The induction time and titer changes of recombinant bacteria GS115 / pPIC9K-PiIFN-α in 5L bioreactor and shake flask culture, respectively; Figure 5The results of SDS-PAGE identification of the recombinant pigeon α-interferon fermentation culture after purification are as follows: Lane 1 is the pPIC9K empty vector control, Lane 2 is the purified PiIFN-α protein, and M is the pre-stained protein molecular weight marker. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0032] Example 1: Preparation of recombinant pigeon α-interferon 1. Optimization and artificial synthesis of pigeon interferon α gene Based on the sequence information of the pigeon interferon α gene (GenBank accession number: XM_833835.1), the signal peptide of the sequence was removed and the sequence was optimized according to the codon preference of Pichia pastoris. The optimized sequence is shown in SEQ ID No.1, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO:2.

[0033] EcoRI and NotI restriction sites were added to both ends of the optimized target gene, and the gene was then inserted into the EcoRI and NotI cloning sites of plasmid pPIC9K. The resulting recombinant plasmid was named Ppic9K-PiIFN-α. This recombinant plasmid was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0034] 2. Construction of recombinant pigeon α-interferon expression engineered strain 2.1 Experimental Methods (1) Linearization of plasmid: The recombinant eukaryotic expression plasmid pPIC9K-PiIFN-α was linearized by single-site digestion with restriction endonuclease Sac I.

[0035] (2) Plasmid transformation and screening of positive transformants: Take 5 μg of linearized plasmid vector, add 80 μl of Pichia pastoris GS115 competent cells, transfer to a pre-cooled 0.2 cm electroporation cuvette, mix well and incubate on ice for 5 min; place the electroporation cuvette on an electroporator for electroporation transformation. Electroporation conditions: voltage 1.5 KV, capacitance 25 μF, resistance 200 Ω.

[0036] Immediately after electroporation, add 1 ml of pre-cooled 1M sorbitol and transfer to a 1.5 ml EP tube. Incubate at 30°C for 1 h. Take 200 μl and spread it onto YPD plates containing 0.5 mg / ml, 0.75 mg / ml, 1 mg / ml, and 2 mg / ml genimycin, respectively. Incubate at 30°C for 3-5 days. High-copy strains are screened by different concentrations of resistance.

[0037] 2.2 Experimental Results Positive transformants were identified by PCR, using the following specific method: Pick a single colony growing on a YPD plate, resuspend it in 10 μL of sterile water, add 1 U of lysozyme litycase, incubate at 30°C for 10 min, freeze at -80°C for 10 min, and then separate the supernatant.

[0038] Using the supernatant obtained above as a DNA template, PCR was performed. The following 20ul PCR reaction system was set up: 10ul of 2×Hieff PCR Master Mix, 1ul of 5'AOX1 primer, 1ul of 3'AOX1 primer, 2ul of DNA template, and 6ul of sterile water.

[0039] The PCR procedure is as follows: 95℃ for 5 minutes 94℃ for 1 minute 54℃ for 1 minute 72℃ for 1 minute 72℃ for 7 minutes, 30 cycles; After amplification, 10 μL was used for agarose gel electrophoresis, yielding two bands of approximately 2.2 kb and 1.0 kb, with the 1.0 kb band being the target band. The results demonstrated the successful screening of high-copy-positive transformants with the Mut+ phenotype, with a positive rate of 99% for His+Mut+ transformants from pPIC9K-Pi-IFN. Subsequent sequencing confirmed the absence of any issues.

[0040] 3. Preparation of recombinant pigeon α-interferon by shake-flask culture 3.1 Experimental Methods The recombinant pigeon α-interferon expression strain prepared in Example 2 was inoculated into 5 mL of YPD culture medium. The next day, 1.0 mL of the strain was inoculated into 50 mL of BMGY culture medium and cultured at 30°C and 250 rpm for 16-18 h. The following day, 5% of the culture medium volume was added to 200 mL of BMMY liquid culture medium for resuspension and cultured at 28°C and 250 rpm. During the induction phase, methanol was added every 24 hours to a final concentration of 1%. After 96 hours of induction, the supernatant was collected by centrifugation at 8000 rpm for 30 min. The molecular weight and purity of the recombinant protein were analyzed by SDS-PAGE and Western blot.

[0041] 3.2 Experimental Results and Analysis The theoretical relative molecular mass of recombinant pigeon α-interferon is 20 kDa, but in actual expression, yeast cells have glycosyltransferases and glycosylation enzymes, which can perform N- and O-glycosylation during protein folding and modification, thus increasing the mass of recombinant pigeon α-interferon.

[0042] The results of Western blot identification of the supernatant are as follows: Figure 2 As shown, the reagent size of the recombinant pigeon α-interferon expressed by the yeast cells is about 30 kDa, and the purity of the recombinant pigeon interferon in the fermentation broth reaches 98%.

[0043] Example 2: Optimization of shake-flask fermentation conditions for recombinant pigeon α-interferon engineered strain 1. Experimental Methods Multiple groups were set up to prepare recombinant pigeon α-interferon under different fermentation conditions according to the method of Example 3, in order to optimize the induction conditions such as pH, methanol concentration, and induction temperature during fermentation. Other conditions and operating methods for each group were the same as in Example 3.

[0044] (1) Optimization of induction pH: Four fermentation systems with induction pH (pH5.0, pH5.5, pH6.0, pH6.5) were set up. 1% methanol was added every 24 hours. After 96 hours, samples were taken to test the titer. The optimal induction pH was determined based on the titer test results.

[0045] (2) Methanol concentration optimization: Expression was induced with methanol concentrations of 0.5%, 1% and 1.5% respectively. After 96 hours of induction, the optimal methanol concentration was determined based on the titer results.

[0046] (3) Optimization of induction temperature: 25℃, 28℃ and 30℃ were selected as induction temperatures respectively. Fermentation was stopped after 96h and the activity was measured. The optimal induction temperature was determined based on the titer test results. The titer test method is the same as that in Example 8.

[0047] 2. Experimental Results and Analysis The results in Table 1 show that the optimal conditions for shake-flask fermentation of recombinant pigeon α-interferon are: induction pH 6.0, induction at 28℃, and addition of 1% methanol (v / v) every 24 h. Subsequent fermentation in a 5L reactor will be used to verify these optimal conditions.

[0048] Table 1 Results of the optimization experiment of shake flask fermentation conditions

[0049] Example 3: Fermentation method of recombinant pigeon α-interferon engineered strain in a 5L reactor This embodiment provides an optimized 5L reactor fermentation process for recombinant pigeon α-interferon engineered strains. The specific method is as follows: 1. Experimental Methods (1) Preparation of seed liquid: Engineered yeast expressing recombinant pigeon α-interferon was inoculated into YPD culture medium and cultured at 30℃ and 250 r / min for 16-18 h. The OD was measured the next day using a UV spectrophotometer. 600 .

[0050] (2) Growth stage: 2.0L of BSM medium was inoculated into a 5L reactor, and seed culture was inoculated into the reactor at a volume ratio of 5%. The rotation speed and aeration rate were dynamically adjusted to maintain dissolved oxygen at 25%-40%. Ammonia was used to maintain the pH at 6.0±0.05. The culture temperature was set at 30℃. The culture time was 20h-23h.

[0051] (3) Glycerin feeding stage: When the DO curve rises rapidly in a short period of time, it indicates that the glycerol in the reactor has been consumed. Then, feed is started with 50% (w / v) glycerol containing 5‰ PTM1 at a feed rate of 16 mL / h / L. After 4 hours of incubation, feed is stopped and the reactor is starved for 45-60 minutes until the glycerol in the tank is depleted.

[0052] (4) Induction of expression stage: The dissolved oxygen (DO) was maintained above 18% by adjusting the rotation speed and aeration rate, the temperature was set at 28℃, and the pH of the fermentation broth was maintained at 6.0±0.05 by using ammonia.

[0053] The initial induction phase was the methanol adaptation phase, with an initial methanol flow rate of 5.0 mL / h / L. After 24 h of induction, methanol was added at a flow rate of 6.0 mL / h / L. After 48 h of induction, methanol was added at a flow rate of 7.0 mL / h / L. After 72 h of induction, methanol was added at a flow rate of 8.0 mL / h / L until fermentation ended at 78 h.

[0054] Both the methanol and glycerol feeds contain 10‰ PTM1 to supplement trace elements.

[0055] The changes in yeast density and the potency of recombinant pigeon α-interferon were compared between the shake flask culture medium prepared in Example 3 and the fermentation broth prepared in this example at different induction times.

[0056] 2. Experimental Results and Analysis As shown in Table 2 and Figure 3 As shown in Table 3, the yeast density obtained by the reactor fermentation method in this embodiment at different induction times was significantly higher than that obtained by the shake flask culture method; Figure 4 As shown, the recombinant pigeon α-interferon obtained by the reactor fermentation method in this embodiment has a potency that is at least three orders of magnitude higher than that obtained by shake flask culture.

[0057] Table 2 Comparison of yeast density between the two culture methods

[0058] Table 3 Comparison of product titers between the two culture methods

[0059] Example 4: Optimization of induction conditions for fermentation of recombinant pigeon α-interferon engineered strain in a 5L reactor. This embodiment optimizes the conditions for the induction expression stage of step (4) in the 5L reactor fermentation process.

[0060] This embodiment attempts and optimizes two methods: 1. Scheme (1): During the induction expression stage, the fermentation broth was maintained at a pH of 6.0±0.05 by adjusting the rotation speed to 550 rpm and setting the temperature to 28℃, and by using ammonia water. After parameter adjustment, methanol was added in a controlled flow at 3-8 mL / h / L to maintain a DO fluctuation core area ≥20 until fermentation ended. Samples were taken during the induction expression stage for testing, and the results are as follows:

[0061] The potency can reach 10 after the can is placed in the jar using a step-controlled method. 6.5 U / ml, there is still room for improvement through optimization.

[0062] 2. Scheme (2): Induction expression stage, the fermentation speed was adjusted to 550 rpm, the temperature was set to 28℃, and methanol was added at fermentation volume ratios of 0.5%, 1%, and 1.5% until fermentation was completed. Samples were taken during the induction expression stage for testing, and the results are as follows: ① Add methanol at a volume ratio of 0.5%

[0063] ② Add methanol at a volume ratio of 1%.

[0064] ③ Add methanol at a volume ratio of 1.5%.

[0065] Through three experiments, the sample titers obtained by the feed-on method based on the fermentation volume ratio were not ideal. Considering the two feed-on methods, the first feed-on method will be optimized in the future.

[0066] 3. Scheme (3): Optimization of methanol feeding: 1) Taking the initial induction as an example, five experimental groups were set up with the following differences: the flow acceleration rates were 3.5, 4.0, 4.5, 5.0 and 5.5 mL / h / L, respectively. Other conditions were the same as in Example 3. The potency was detected by taking samples after 24 hours. The potency of the samples is shown in the figure. The potency was highest in the range of 5.0±0.5 mL / h / L. Therefore, it was determined that the flow acceleration rate of methanol could be within 5.0±0.5 mL / h / L after 24 hours of induction.

[0067]

[0068] 2) Five experimental groups were set up during the 24h induction period, with the following differences: the flow rates were 4.5, 5.0, 5.5, 6.0 and 6.5 mL / h / L, respectively. Other conditions were the same as in Example 3. The potency of the samples was tested after 48h. The potency of the samples is shown in the table below. The potency was highest in the range of 6.0±0.5 mL / h / L. Therefore, the flow rate of methanol was determined to be within 6.0±0.5 mL / h / L during the 24h induction period.

[0069]

[0070] 3) Five experimental groups were set up for 48h of induction, with the following differences: the flow rates were 5.5, 6.0, 6.5, 7.0 and 7.5 mL / h / L, respectively. Other conditions were the same as in Example 3. The potency was detected by taking samples after 72h. The potency of the samples is shown in the table below. The potency was highest in the range of 7.0±0.5 mL / h / L. Therefore, it was determined that the flow rate of methanol could be within 7.0±0.5 mL / h / L after 48h of induction.

[0071]

[0072] 4) Five experimental groups were set up for 72h of induction, with the following differences: the flow rates were 7.0, 7.5, 8.0, 8.5 and 9.0 mL / h / L, respectively. Other conditions were the same as in Example 3. The potency was tested by taking samples at 96h. The potency of the samples is shown in the table below. The potency was highest in the range of 8.0±0.5 mL / h / L. Therefore, it was determined that the flow rate of methanol could be within 8.0±0.5 mL / h / L for 72h of induction.

[0073]

[0074] 4. Scheme (4): DO optimization control: Based on the above optimized conditions, experiments were conducted in two groups. The difference between the two groups was whether high-purity oxygen was used during the recombinant protein induction expression process to maintain DO within a certain range (12%-55%). Other conditions were the same as in Example 3. Finally, samples were taken after 78 hours of induction for titer testing, and the group with the higher titer was selected as the optimal fermentation condition.

[0075] The sample titers are shown in the table below. These results indicate that using high-purity oxygen during the recombinant protein induction expression process can improve the product titer.

[0076]

[0077] Based on the above exploration conditions, the optimal conditions for the induction expression stage of the 5L reactor fermentation process for the recombinant pigeon α-interferon engineered strain were finally determined to be: an initial methanol flow rate of 5.0±0.5 mL / h / L; a methanol flow rate of 6.0±0.5 mL / h / L after 24 h of induction; a methanol flow rate of 7.0±0.5 mL / h / L after 48 h of induction; and a methanol flow rate of 8.0±0.5 mL / h / L after 72 h of induction. Under these conditions, the recombinant pigeon α-interferon exhibited the highest potency. Oxygen regulation during the induction process significantly increased the expression level of the recombinant pigeon α-interferon.

[0078] Example 5: Purification of Recombinant Pigeon Alpha Interferon This embodiment uses a self-assembled 5ml nickel column kit, and the purification reagent used is the His-tagged protein purification kit from Beyotime.

[0079] 1. Purification method: Take 200 ml of the fermentation broth prepared in Example 3 and centrifuge at 10,000 rpm for 20 minutes at 4°C. Collect the supernatant and prepare to purify the recombinant pigeon α-interferon through a nickel column.

[0080] Before using a nickel column, it needs to be equilibrated. Add 4 column volumes of deionized water to the nickel column for washing, and then add 8 column volumes of Binding-beffer to equilibrate the nickel column. After equilibration, load the sample.

[0081] The sample was loaded at a flow rate of 10 column volumes / hour, and the flow-through was collected. The nickel column was then washed with 15 column volumes of BindingBeffer to remove contaminating proteins. Proteins were eluted with an appropriate amount of 200 mM imidazole. Protein purity was analyzed using a 12% SDS-PAGE gel.

[0082] The purified recombinant pigeon α-interferon was analyzed using a BCA protein assay kit (Solepro BCA protein concentration assay kit, product number: PC0020), and then filtered through a 0.22 μm filter for sterilization and stored at -80℃ for later use.

[0083] The fermentation broth prepared by the shake-flask fermentation method in Example 2 was purified according to the method described in this example and used as a control group.

[0084] 2. Experimental Results and Analysis SDS-PAGE identification results are as follows: Figure 5 As shown, the purity of recombinant pigeon α interferon in the purified product is high; the results of BCA protein quantification analysis are shown in Table 4. Compared with shake flask culture, the protein content of recombinant PiIFN-α obtained by reactor fermentation method of scale-up culture is significantly improved. After 78 hours of culture, the yield of the target protein is about 10 times higher than that of the target protein content of shake flask culture.

[0085] Table 4 shows the quantitative analysis of BCA protein after purification of recombinant pigeon interferon PiIFN-α.

[0086] Example 7: Bioactivity Detection of Recombinant Pigeon Alpha Interferon The bioactivity of the culture supernatant from the shake-flask culture in Example 2 and the recombinant pigeon α-interferon prepared and purified by the reactor fermentation method in Example 3 were tested according to the following methods, and the activities of the recombinant pigeon α-interferon prepared by the two methods were compared.

[0087] 1. Experimental Method: The antiviral activity of recombinant pigeon alpha interferon was determined using the microcytopathic effect inhibition method on the CEF-VSV system (chicken embryo fibroblast-vesicular stomatitis virus), and the preparation of the test solution was also performed. Discard the culture medium from the flask containing a confluent CEF (chicken embryo fibroblast) cell monolayer. Wash three times with PBS, digest with 0.125% trypsin, and collect the cells. Dilute the cells with culture medium to a concentration of 2 × 10⁶ cells per milliliter using cell counting. 5 -3×10 5 Cell suspensions of 100 μl were seeded into 96-well culture plates and cultured at 37°C and 5% CO2 for 24 hours.

[0088] The following day, the alpha interferon from the pigeons to be tested was serially diluted 10-fold, from 10... -1 -10 -8 Eight dilutions were prepared, with four replicates for each dilution. The supernatant in the cell culture plate was discarded, and the diluted sample was added to the plate and incubated at 37°C and 5% CO2 for 16 hours. The prepared vesicular stomatitis virus (VSV) viral solution (stored at -80°C) was diluted to 100 TCID50 with cell maintenance medium. 50After adding 0.1 mL, add 100 μl to each well of the culture plate and place it in a 37°C, 5% CO2 incubator. Cell control and positive control are also set up.

[0089] After culturing for 18–24 hours, cytopathic effects were observed and the median number of cytopathic effects was calculated. The highest dilution showing 50% cytopathic effects was defined as one interferon unit (U). The activity (U / mL) of the pigeon α-interferon recombinant protein was calculated using the Reed-Muench method (median effective dose cumulative method), and the activity was used to determine the antiviral activity of the pigeon α-interferon recombinant protein.

[0090] 2. Experimental Results and Analysis The results showed that the antiviral activity of pigeon alpha interferon in shake flasks was 10. 5.5 U / mL (enzyme activity, i.e., potency), which is 3.16 × 10⁻⁶. 5 Its specific activity is 3.16 × 10 U / mL. 5 ×10 3 U / L / 86mg / L=3.67×10 6 U / mg; The pigeon α-interferon cultured in the reactor exhibited the highest antiviral activity (3.16 × 10⁻⁶ h after induction). 8 U / mL, compared to shake flask culture, reactor culture increased the titer of pigeon α-interferon by 1000 times (10 U / mL). 8.5 / 10 5.5 ).

[0091] In summary, the recombinant pigeon α-interferon obtained by reactor culture in this invention has good antiviral activity, maintains its viability for a long time under high-density growth conditions, and has low culture medium component costs, making it suitable for large-scale production and application.

[0092] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A recombinant pigeon interferon α gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A recombinant expression vector carrying the pigeon interferon α gene, characterized in that, It is formed by recombination of an expression vector and the recombinant pigeon interferon α gene as described in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that, The expression vector is a pPIC9K series plasmid.

4. A recombinant host bacterium, characterized in that, The recombinant host cell is transformed into the recombinant expression vector as described in claim 2 or 3.

5. The recombinant host bacterium according to claim 4, characterized in that, The recombinant host strain used is Pichia pastoris cells.

6. A method for preparing recombinant pigeon α-interferon, characterized in that, Includes the following steps: The recombinant host bacteria as described in claim 4 were fermented and cultured, and the supernatant of the fermentation broth was collected and purified to obtain recombinant pigeon α-interferon.

7. The method for preparing recombinant pigeon α-interferon according to claim 6, characterized in that, The fermentation culture method includes the following steps: (1) Growth stage: Add 1~2.5L of fermentation medium to the fermentation equipment, and then inoculate the seed liquid of the recombinant host bacteria as described in claim 4 into the fermentation system. The aeration rate is 3-6L / min, the rotation speed is 250-650 rpm, the temperature is 28-30℃, the dissolved oxygen content is maintained at 20%-50% by micro-oxygenation, and the pH is maintained at 6.0±0.

05. (2) Glycerol feeding stage: When the culture is about 24h-28h, add 50% glycerol containing 1-8‰ PTM1 by mass volume at a rate of 13-18mL / h / L, continue to culture for 3-5h, stop feeding, and when the glycerol in the tank is exhausted, starve culture for 45-90min; (3) Induction expression stage: The pH was adjusted to 6.0±0.05, and methanol was added in stages at a flow rate of 5 mL / h / L~10 mL / h / L to induce fermentation. The dissolved oxygen content was maintained at 12%-55% by dynamically adjusting the rotation speed and aeration rate until the fermentation ended after 78 hours. 1-8‰ PTM1 was added to the methanol to supplement trace elements.

8. The method for preparing recombinant pigeon α-interferon according to claim 7, characterized in that, The specific methods for the induction expression phase are as follows: The pH was adjusted to 6.0±0.05, methanol was added, and induction was started at a flow rate of 5.0±1.0 mL / h / L for the initial fermentation medium. The dissolved oxygen level was maintained at 12%-55% by dynamically adjusting the aeration rate and the rotation speed. After 24 h of induction, methanol was added at a flow rate of 6.0±1.0 mL / h / L. After 48 h of induction, methanol was added at a flow rate of 7.0±1.0 mL / h / L. After 72 h of induction, methanol was added at a flow rate of 8.0±1.0 mL / h / L until fermentation ended at 78 h.

9. The method for preparing recombinant pigeon α-interferon according to claim 6, characterized in that, The method for constructing the recombinant host bacterium is as follows: The pigeon interferon α gene with the nucleotide sequence shown in SEQ ID NO.1 was cloned into an expression vector to construct a recombinant expression vector, which was then transformed into a host bacterium to obtain a recombinant host bacterium.

10. The application of recombinant pigeon α-interferon prepared by the method of any one of claims 6 to 8 in determining its antiviral activity in a chicken embryo fibroblast-vesicular stomatitis virus system.

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