阅读说明：本技术 一种猪α-干扰素的发酵工艺 (Fermentation process of porcine alpha-interferon ) 是由 郭海岩 刘刚 王兴业 韩国英 苗玉和 韩法杰 王红军 孙秀丽 于 2019-11-01 设计创作，主要内容包括：本发明涉及猪干扰素的发酵生产,具体涉及一种猪α-干扰素的发酵工艺。该工艺包括菌株活化及筛选、一级种子培养、二级种子培养和发酵培养；所述菌株为含有重组猪α-干扰素表达载体的甲醇酵母；所述发酵培养包括菌体快速生长阶段、菌体边富集边诱导阶段和菌体诱导阶段；在菌株活化及筛选中,通过递增培养基中的抗生素浓度对所述菌株进行至少三次抗性筛选,得到稳定的多拷贝菌株；在菌体边富集边诱导阶段,向诱导培养基中流加甲醇和甘露醇溶液；在菌体诱导阶段,向诱导培养基中流加甲醇；所述诱导培养基中添加有亮氨酸和油酸。采用本发明的发酵工艺生产猪α-干扰素,产量高达21.6g/L,发酵历时仅54～60h。(The invention relates to fermentation production of porcine interferon, in particular to a fermentation process of porcine alpha-interferon. The process comprises strain activation and screening, primary seed culture, secondary seed culture and fermentation culture; the strain is methanol yeast containing a recombinant porcine alpha-interferon expression vector; the fermentation culture comprises a thallus rapid growth stage, a thallus enrichment and induction stage and a thallus induction stage; in the activation and screening of the strain, the strain is subjected to resistance screening for at least three times by increasing the concentration of antibiotics in a culture medium to obtain a stable multi-copy strain; in the stage of enriching and inducing the thalli, adding methanol and mannitol solution into an induction culture medium; in the thallus induction stage, methanol is added into an induction culture medium; leucine and oleic acid are added into the induction culture medium. The fermentation process of the invention is adopted to produce the porcine alpha-interferon, the yield is up to 21.6g/L, and the fermentation time is only 54-60 h.)

1. A fermentation process of porcine alpha-interferon is characterized by comprising the following steps: comprises strain activation and screening, primary seed culture, secondary seed culture and fermentation culture; the strain is methanol yeast containing a porcine alpha-interferon expression vector; the fermentation culture comprises a thallus rapid growth stage, a thallus enrichment and induction stage and a thallus induction stage;

in the strain activation and screening, performing resistance screening on the strain for at least three times by increasing the antibiotic concentration in a culture medium to obtain a stable multi-copy strain;

in the step of enriching and inducing the thalli, adding methanol and mannitol solution into an induction culture medium; in the thallus induction stage, methanol is added into an induction culture medium in a flowing mode;

leucine and oleic acid are added into the induction culture medium.

2. The fermentation process of claim 1, wherein: the expression vector contains a bleomycin resistance gene; in the strain activation and selection, the strain is inoculated into a culture medium containing 300 mu g/ml, 600 mu g/ml and 900 mu g/ml bleomycin in sequence, and three times of resistance selection are carried out.

3. The fermentation process of claim 1, wherein: the induction medium contained leucine at a final concentration of 1mg/ml and oleic acid at a concentration of 0.02% by volume.

4. The fermentation process of claim 1, wherein: in the step of enriching and inducing the thalli, 0.1g/mL mannitol solution is added into an inducing culture medium at the rate of 6-9 mL/h/L, and methanol is added into the inducing culture medium at the rate of 2-3 mL/h/L; and in the thallus induction stage, methanol is added into an induction culture medium at the speed of 9-12 mL/h/L.

5. The fermentation process of claim 1, wherein: the strain adopts a strain with a preservation number of CCTCCM 2019749.

6. The fermentation process of claim 1, wherein: the rapid growth stage of the thalli is from the beginning of fermentation to the end of the consumption of the initial carbon source in the fermentation broth; the step of enriching and inducing the thallus from the end of the quick thallus growth stage to the wet thallus weight of 400 g/L; the step of inducing the bacteria is performed from the end of the step of inducing the bacteria while enriching to the time when the wet weight of the bacteria reaches more than 500 g/L.

7. The fermentation process of claim 1, wherein: the fermentation culture comprises the following steps:

and (3) a thallus rapid growth stage: adding the secondary seed solution into an induction culture medium, controlling the temperature at 30 ℃ and the pH value at 5.0, keeping the dissolved oxygen at more than 30% by adjusting the rotating speed and the ventilation amount during fermentation, periodically sampling and measuring the surplus of the carbon source, and entering a thallus enrichment-edge induction stage when the initial carbon source is completely consumed;

enrichment and induction of the thalli: supplementing 0.1g/mL mannitol solution at the rate of 6-9 mL/h/L, supplementing methanol at the rate of 2-3 mL/h/L, controlling the rotation speed of 200rpm, the temperature of 28 ℃, the pH value of 5.0 and the dissolved oxygen of 20-30%, sampling at regular time to determine the wet weight of cells, stopping supplementing the mannitol solution when the wet weight of thalli reaches more than 400g/L, and transferring to a thalli induction stage;

and (3) a thallus induction stage: and (3) supplementing methanol at the rate of 9-12 mL/h/L, controlling the rotating speed to be 200rpm, the temperature to be 28 ℃, the pH value to be 6.0 and the dissolved oxygen to be 20-30%, and ending the fermentation when the wet weight of the thalli reaches more than 500 g/L.

8. The fermentation process of claim 2, wherein: the strain activation and screening comprises the following steps:

screening for the first time: dissolving 0.8-1.2 g of strain freeze-dried powder in 1mL of sterile YPD liquid culture medium, taking bacterial liquid, streaking on a YPD solid culture medium containing 300 mu g/mL of bleomycin, and then culturing in a constant-temperature incubator at 30 ℃ for 48 hours;

and (3) screening for the second time: picking single colony from the plate screened for the first time, performing streak culture on YPD solid culture medium containing 600 mug/ml bleomycin, and then placing in a constant temperature incubator at 30 ℃ for culturing for 48 h;

and (3) third screening: single colonies were picked from the second plate and streaked on YPD solid medium containing 900. mu.g/ml bleomycin, followed by incubation in a 30 ℃ incubator for 48 hours.

9. The fermentation process according to any one of claims 1 to 8, wherein: every 100ml of the induction culture medium contains 1g of yeast powder, 2g of tryptone, 2ml of glycerol, 0.34g of YNB and 1.0941gKH₂PO₄、0.4473g K₂HPO₄·3H₂O, 0.1g leucine, 0.02ml oleic acid and 0.1ml biotin solution at 0.4 mg/ml.

10. Use of a fermentation process according to any one of claims 1 to 9 for the production of porcine interferon-alpha.

Technical Field

The invention relates to fermentation production of porcine interferon, in particular to a fermentation process of porcine alpha-interferon.

Background

China is a world pig-raising big country, more than 69382 thousands of pigs are produced in 2018, and billions of yuan is lost to the live pig breeding industry due to outbreak of viral diseases every year. A new product for preventing and treating the porcine viral diseases, which is efficient, safe and free from toxic and side effects, is urgently needed to be searched. The most used porcine leukocyte interferon in the market at present is produced by induced method of Sichuan Honghe Biotechnology limited company, and the new veterinary certificate of Ministry of agriculture [ (97) new veterinary certificate No. 01 ]. At present, escherichia coli and pichia pastoris are used for successfully expressing recombinant interferon, but no recombinant porcine recombinant interferon in China obtains a new veterinary certificate so far, and no imported recombinant porcine interferon obtains an imported registration certificate. The great obstacles are the complicated production process and low yield.

The promoter of Alcohol Oxidase (AOXI) gene of Pichia pastoris has strong inductivity and strong startability, and is a common expression system capable of efficiently expressing foreign proteins, but the expression conditions of different foreign proteins in Pichia pastoris are greatly different, and the fermentation process is one of the important factors causing the difference.

The traditional pichia pastoris fermentation process comprises two processes: a growth-first induction mode and a growth-side induction mode. The growth-first induction mode uses glycerol as a carbon source, the yeast reaches higher density after about 34-48h of growth period, then methanol is added for induction for about 72-120h, the final density of the engineering bacteria can reach about 450g/L generally, and the whole fermentation process lasts for 108 plus 168 h. The process has obvious disadvantages, and is time-consuming and labor-consuming. The mode of growth and induction is to search a balance point between protein expression and thallus production, and does not require to carry out protein induction expression after the thallus reaches a very high density, and simultaneously omits the feed supplement growth period, so the whole production period is short. However, compared with the mode of growth before induction, the process reduces the bacterial density and the yield of foreign proteins.

Disclosure of Invention

In order to make up for the defects in the field, the invention explores a fermentation process suitable for expressing the porcine alpha-interferon in pichia pastoris, and has the advantages of short fermentation time, high yield and the like.

The invention provides a fermentation process of porcine alpha-interferon, which is characterized by comprising the following steps: comprises strain activation and screening, primary seed culture, secondary seed culture and fermentation culture; the strain is methanol yeast containing a porcine alpha-interferon expression vector; the fermentation culture comprises a thallus rapid growth stage, a thallus enrichment and induction stage and a thallus induction stage;

in the strain activation and screening, performing resistance screening on the strain for at least three times by increasing the antibiotic concentration in a culture medium to obtain a stable multi-copy strain;

in the step of enriching and inducing the thalli, adding methanol and mannitol solution into an induction culture medium; in the thallus induction stage, methanol is added into an induction culture medium in a flowing mode;

leucine and oleic acid are added into the induction culture medium.

In some embodiments, the expression vector contains a bleomycin resistance gene; in the strain activation and selection, the strain is inoculated into a culture medium containing 300 mu g/ml, 600 mu g/ml and 900 mu g/ml bleomycin in sequence, and three times of resistance selection are carried out.

In a preferred embodiment, the induction medium contains leucine at a final concentration of 1mg/ml and oleic acid at a volume percent concentration of 0.02%.

In a preferred embodiment, in the induction stage while enriching the thalli, 0.1g/mL of mannitol solution is added into an induction culture medium at the rate of 6-9 mL/h/L, and methanol is added into the induction culture medium at the rate of 2-3 mL/h/L; and in the thallus induction stage, methanol is added into an induction culture medium at the speed of 9-12 mL/h/L.

In a preferred embodiment, the strain adopts a strain with a preservation number of CCTCC M2019749.

In some embodiments, the period from the start of fermentation to the time the initial carbon source in the fermentation broth is depleted is the period of rapid growth of the biomass; the step of enriching and inducing the thallus from the end of the quick thallus growth stage to the wet thallus weight of 400 g/L; the step of inducing the bacteria is performed from the end of the step of inducing the bacteria while enriching to the time when the wet weight of the bacteria reaches more than 500 g/L.

In some embodiments, the fermentation culture comprises the steps of:

and (3) a thallus rapid growth stage: adding the secondary seed solution into an induction culture medium, controlling the temperature at 30 ℃ and the pH value at 5.0, keeping the dissolved oxygen at more than 30% by adjusting the rotating speed and the ventilation amount during fermentation, periodically sampling and measuring the surplus of the carbon source, and entering a thallus enrichment-edge induction stage when the initial carbon source is completely consumed;

enrichment and induction of the thalli: supplementing 0.1g/mL mannitol solution at the rate of 6-9 mL/h/L, supplementing methanol at the rate of 2-3 mL/h/L, controlling the rotation speed of 200rpm, the temperature of 28 ℃, the pH value of 5.0 and the dissolved oxygen of 20-30%, sampling at regular time to determine the wet weight of cells, stopping supplementing the mannitol solution when the wet weight of thalli reaches more than 400g/L, and transferring to a thalli induction stage;

and (3) a thallus induction stage: and (3) supplementing methanol at the rate of 9-12 mL/h/L, controlling the rotating speed to be 200rpm, the temperature to be 28 ℃, the pH value to be 6.0 and the dissolved oxygen to be 20-30%, and ending the fermentation when the wet weight of the thalli reaches more than 500 g/L.

In some embodiments, the strain activation and screening comprises the steps of:

screening for the first time: dissolving 0.8-1.2 g of strain freeze-dried powder in 1mL of sterile YPD liquid culture medium, taking bacterial liquid, streaking on a YPD solid culture medium containing 300 mu g/mL of bleomycin, and then culturing in a constant-temperature incubator at 30 ℃ for 48 hours;

and (3) screening for the second time: picking single colony from the plate screened for the first time, performing streak culture on YPD solid culture medium containing 600 mug/ml bleomycin, and then placing in a constant temperature incubator at 30 ℃ for culturing for 48 h;

and (3) third screening: single colonies were picked from the second plate and streaked on YPD solid medium containing 900. mu.g/ml bleomycin, followed by incubation in a 30 ℃ incubator for 48 hours.

In a preferred embodiment, the induction medium contains 1g of yeast powder, 2g of tryptone, 2ml of glycerol, 0.34g of YNB, 1.0941gKH per 100ml of induction medium₂PO₄、0.4473g K₂HPO₄·3H₂O, 0.1g leucine, 0.02ml oleic acid and 0.1ml biotin solution at 0.4 mg/ml.

The invention also provides application of any one of the fermentation processes in production of the porcine alpha-interferon.

The fermentation process of the porcine alpha-interferon provided by the invention comprises the steps of strain activation and screening, primary seed culture, secondary seed culture and fermentation culture (a thallus rapid growth stage, a thallus enrichment stage and an induction stage). The fermentation process of the invention is adopted to produce the porcine alpha-interferon, the yield is up to 21.6g/L, and the fermentation process lasts for 54-60 hours.

Compared with the traditional fermentation process of the porcine alpha-interferon, the invention has the main improvements that:

(1) the invention inoculates the strains on YPD culture medium containing 300 mug/ml, 600 mug/ml and 900 mug/ml bleomycin in sequence before fermentation, and screens out stable multi-copy high-yield strains for fermentation, thereby improving the yield of the pig alpha-interferon.

(2) The invention adds leucine and oleic acid into the induction culture medium, thereby improving the yield of the porcine alpha-interferon. Leucine is amino acid with higher proportion in the pig alpha-interferon sequence, accounts for 14.20 percent of the total amount of the amino acid, and can supplement the deficiency of leucine caused by the mass expression of the pig alpha-interferon; oleic acid has a strong promotion effect on an AOX1 promoter, can accelerate methanol metabolism and improve the utilization rate of methanol.

(3) The invention carries out methanol feeding twice on the basis of enriching the thalli. The first feeding is that in the phase of enrichment and induction of the thalli, methanol and mannitol are mixed and fed, and the thalli is enriched and the expression of the pig alpha-interferon is induced; the second feeding is in the thallus inducing stage, and only methanol is used for feeding. Compared with the traditional fermentation process of growth and induction, the fermentation time is shortened by more than 30%, compared with the traditional fermentation process of growth and induction, the initial induction bacterial quantity is improved, and the pig alpha-interferon yield is increased by 3-5 times.

The invention preferably adopts the strain with the preservation number of CCTCC M2019749, and the strain is constructed by people. A protein sequence of the porcine alpha-interferon is found on an NCBI website (https:// www.ncbi.nlm.nih.gov /), the original signal peptide of the porcine alpha-interferon is deleted, the amino acid with degenerate codons is statistically analyzed, and the codon with high use frequency is selected to obtain a new recombinant porcine alpha-interferon gene sequence. Through gene synthesis, a new recombinant porcine alpha-interferon gene is obtained, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1. The new gene is inserted into a secretory multicopy pPICZaA expression vector and is transfected into pichia pastoris, and a high-concentration bleomycin-containing flat plate is adopted for screening to obtain the pichia pastoris with obviously improved recombinant pig alpha-interferon secretion capacity, and the strain preservation is carried out. The porcine alpha-interferon produced by adopting the preserved strain and the fermentation process has the advantages of high yield of 21.6g/L, fermentation duration of only 54-60 h and extremely high application value.

The engineering bacteria for producing the porcine alpha-interferon provided by the invention have the following preservation information:

the preservation name is: pichia pastoris X33/PoIFN-alpha

And (3) classification and naming: pichia sp.X33/PoIFN-alpha

The preservation date is as follows: 9/25/2019

The preservation number is: CCTCC M2019749

The preservation unit: china center for type culture Collection

The address of the depository: china center for type culture Collection, Taiwan university, eight Wuhan universities in flood and mountain areas, Wuhan city, Hubei province, zip code: 430072, E-mail: cctcc @ whu. edu. cn.

Drawings

FIG. 1 is a plasmid map of pPICZ alpha A-porcine alpha interferon constructed in the present invention;

FIG. 2 shows the electrophoretogram of Pichia pastoris screened to contain the recombinant porcine alpha-interferon gene of the invention.

Detailed Description

The present invention is described in detail below with reference to examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.

Experimental reagent:

DNA purification recovery kit, 2 XPCR Master Mix, restriction enzymes EcoR I and Not I, 10 XPuffer, T4-DNA ligase, D-sorbitol, yeast extract, yeast powder, tryptone, peptone, YNB, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, biotin, bleomycin, glycerol, sodium chloride, ammonium sulfate, glucose were purchased from Biotechnology (Shanghai) GmbH;

pPICZ α A plasmid: purchased from Shanghai Xinyu Biotechnology, Inc., under item number XY 0216;

DH5 α competent cells: purchased from Invitrogen, cat # 18265017;

x-33 Yeast competent cells: purchased from Invitrogen, cat # C18000;

leucine: purchased from tequila biotech ltd;

oleic acid: purchased from Huainan Quanrun environmental protection science and technology Co., Ltd;

mannitol: purchased from Guangdong Guanghua science and technology, Inc.;

methanol: purchased from Qingdao brochure chemical ltd;

cytolytic enzymes: purchased from sigma corporation, usa;

plasmid extraction and purification kit: purchased from Qiagen corporation;

the pig alpha interferon quantitative detection kit comprises: purchased from Jiangsu Kejing Biotech, Inc.;

0.4mg/ml biotin solution: 0.04g of biotin was weighed, dissolved in 100mL of single distilled water, and sterile-filtered through a cellulose filter with a pore size of 0.22 μm.

Culture medium:

LB Medium

Each 100ml of LB medium contained: 1g tryptone, 0.5g yeast extract, 1g sodium chloride, pH 7.4.

The preparation method comprises the following steps: at 950ml ddH₂Dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride in O, adjusting pH to 7.4 with NaOH, and adding ddH₂And O is metered to 1L. If a solid medium is prepared, 15g of agar per liter are added. Sterilizing with high pressure steam at 121 deg.C for 20 min.

YPD medium

The formula is as follows: yeast cream: peptone: glucose was 1:2:2 (mass ratio).

The preparation method comprises the following steps: dissolving 10g of yeast extract, 20g of peptone in 900ml of ddH₂In O, 20g of agar is added if the plate is prepared; sterilizing with high pressure steam at 121 deg.C for 20 min; after cooling, 100ml of a separately sterilized (autoclaved at 115 ℃ C. for 15min) glucose solution (containing 20g of glucose) was added.

BMGY medium

Per 100ml of BMGY medium contained: 1g of yeast powder, 2g of tryptone, 1ml of glycerol, 0.34g of YNB (ammonium sulfate and amino acid excluded), 1g of ammonium sulfate, and 1.0941g of KH₂PO₄、0.4473g K₂HPO₄·3H₂O, 0.1ml of 0.4mg/ml biotin solution.

The preparation method of the 100LBMGY culture medium comprises the following steps: in ddH₂Adding 1000g yeast powder, 2000g tryptone, 1L glycerol, 340gYNB (without ammonium sulfate and amino acid), 1000g ammonium sulfate, KH₂PO₄1094.1g，K₂HPO₄·3H₂Heating, stirring and dissolving O447.3 g, fixing the volume to 99900ml, and then sterilizing for 20min by high-pressure steam at 121 ℃; cooling to room temperature, adding 100ml of sterilized biotin solution, mixing, and packaging in sterile container.

BMMY medium

Per 100ml of BMMY medium: 1g of yeast powder, 2g of tryptone, 0.5ml of methanol, 0.34g of YNB (ammonium sulfate and amino acid excluded), 1g of ammonium sulfate, and 1.0941g of KH₂PO₄、0.4473g K₂HPO₄·3H₂O, 0.1ml of 0.4mg/ml biotin solution.

The preparation method of the 1000LBMMY culture medium comprises the following steps: in ddH₂In O is added10000g yeast powder, 20000g tryptone, 5L methanol, 3400g YNB (ammonium sulfate and amino acid free), 10000g ammonium sulfate, KH₂PO₄10941g，K₂HPO₄·3H₂O4473g, heating, stirring to dissolve, diluting to 999900ml, and sterilizing with high pressure steam at 121 deg.C for 20 min; cooling to room temperature, adding 1L biotin solution sterilized separately, mixing, and packaging in sterile container.

Induction medium

Per 100ml induction medium contained: 1g of yeast powder, 2g of tryptone, 0.5ml of methanol, 0.34g of YNB (ammonium sulfate and amino acid excluded), 1g of ammonium sulfate, and 1.0941g of KH₂PO₄、0.4473g K₂HPO₄·3H₂O, 0.1g leucine, 0.02ml oleic acid, 0.1ml biotin solution at 0.4 mg/ml.

The preparation method of 1000L induction medium comprises the following steps: in ddH₂Adding yeast powder 10000g, tryptone 20000g, methanol 5L, YNB 3400g (without ammonium sulfate and amino acid), ammonium sulfate 10000g, and KH₂PO₄10941g，K₂HPO₄·3H₂O4473g, leucine 1000g and oleic acid 200ml are heated, stirred and dissolved, the volume is fixed to 999900ml, and then the high-pressure steam sterilization is carried out for 20min at 121 ℃; cooling to room temperature, adding 1L biotin solution sterilized separately, mixing, and packaging in sterile container.

The experimental reagents which are not particularly described in the invention are all conventional reagents in the field, and can be prepared according to the conventional method in the field or obtained commercially; the experimental procedures not specifically described are conventional in the art and may be referred to, for example, in the Molecular cloning laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual, 2001), or the manufacturer's instructions.