CN111455006B

CN111455006B - Recombinant chicken interferon alpha product expressed by escherichia coli and preparation method and application thereof

Info

Publication number: CN111455006B
Application number: CN202010280783.9A
Authority: CN
Inventors: 王连民; 高应瑞; 刘珂飞
Original assignee: Tianjin Shengji Group Co Ltd
Current assignee: Tianjin Shengji Group Co Ltd
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2021-09-07
Anticipated expiration: 2040-04-10
Also published as: CN111455006A

Abstract

The invention discloses a recombinant chicken interferon alpha product expressed by escherichia coli and a preparation method and application thereof, belonging to the technical field of biology. The invention carries out fermentation culture on recombinant escherichia coli engineering bacteria, adds IPTG into the recombinant escherichia coli engineering bacteria, finishes fermentation after induction for 4-6 hours, obtains bacteria by centrifugation, obtains chicken interferon alpha protein inclusion bodies by crushing and cracking the bacteria, denaturalizes and dissolves the chicken interferon alpha protein inclusion bodies, carries out chromatography purification by using a nickel-agarose gel FF chromatographic column, then carries out protein renaturation, mixes the chicken interferon alpha protein inclusion bodies with a protective agent, simultaneously adds water for injection for dilution, and obtains a finished product of the recombinant chicken interferon alpha by split charging.

Description

Recombinant chicken interferon alpha product expressed by escherichia coli and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant chicken interferon alpha product expressed by escherichia coli, and a preparation method and application thereof.

Background

Interferons are glycoproteins with antiviral activity on the same cell, and achieve prophylactic and therapeutic effects by inhibiting the replication of viral DNA and RNA. Since ISaacs and Lindenmann discovered interferons in 1957, interferons have shown extremely strong antiviral and immunomodulatory activities and broad application prospects. Thus, the research of interferons is receiving more and more attention. Compared with the traditional health care and treatment medicines, the interferon has great superiority in the aspects of treatment effect and use timing.

Because of its powerful antiviral effect, many studies have been reported for treating viral diseases in poultry. A large number of research results show that the recombinant chicken interferon alpha has obvious antiviral effect when used for preventing and treating viral diseases such as chicken avian influenza, infectious bursal disease, infectious bronchitis and the like.

The gene engineering interferon is obtained by subcloning natural interferon gene in animal body into prokaryotic expression vector, transferring into receptor cell, and constructing engineering bacteria. The high-purity interferon product is finally obtained through the steps of engineering bacteria fermentation expression, separation and purification and the like.

The invention discloses a preparation method and application effect of a recombinant chicken interferon alpha product expressed by escherichia coli engineering bacteria, belonging to a genetic engineering biological product obtained by a molecular biological method. The newly designed chicken interferon-alpha gene sequence is recombined to a pET21a (+) vector and then transferred into a receptor escherichia coli through a transformation mode. And an escherichia coli expression system is adopted to express the exogenous gene, which is beneficial to the commercial production of the chicken interferon-alpha gene. The prepared product can be used for preparing disease prevention and treatment medicines and immunopotentiators, and can also be used for theoretical research of poultry immunity mechanism.

Disclosure of Invention

The invention aims to solve the problem of providing a recombinant chicken interferon alpha product expressed by escherichia coli engineering bacteria, a preparation method and application thereof.

The escherichia coli engineering bacteria take BL21(DE3) as a host, and the constructed engineering strains are as follows: coli BL21(DE3)/pET21a (+) -ChIFN alpha. The engineering strain and the construction method thereof are derived from the invention patent 'recombinant chicken interferon alpha' with the application number of 201410755900.7 and the preparation method thereof.

The cDNA sequence of the recombinant chicken interferon alpha expressed by the engineering strain is an optimized gene sequence shown as SEQ ID NO: 1 is shown.

In order to solve the problems, the invention provides a preparation method of a recombinant chicken interferon alpha product expressed by escherichia coli engineering bacteria, which comprises the following steps:

carrying out fermentation culture on escherichia coli engineering bacteria, adding IPTG (isopropyl-beta-thiogalactoside) into the escherichia coli engineering bacteria, finishing fermentation after inducing for 4-6 hours, centrifuging to obtain bacteria, crushing and cracking the bacteria to obtain recombinant chicken interferon alpha protein inclusion bodies, carrying out denaturation and dissolution on the recombinant chicken interferon alpha protein inclusion bodies, carrying out chromatography purification by using a nickel-sepharose FF chromatography column, carrying out protein renaturation, mixing with a protective agent, adding water for injection to dilute, and subpackaging to obtain a finished product of the recombinant chicken interferon alpha;

the renaturation liquid used in the protein renaturation comprises the following components: 0.5M arginine, 2mM EDTA, 0.8mM GSSG (oxidized glutathione), 50mM Tris, 20% glycerol, balance water, pH 7.0. (% by mass)

Preferably, the elution buffer used in the chromatographic purification consists of: 6M GU-HCl, 20mM tris-HCl, 500mM imidazole, balance water, pH 7.5.

Preferably, the engineering bacteria fermentation culture step is as follows: the engineering bacteria are activated and then inoculated in a fermentation medium according to the inoculation amount of 5-10 percent, fermented at the temperature of 36.8-37.2 ℃, the pH value is controlled to be 6.8-7.2, and the dissolved oxygen is controlled to be more than or equal to 30 percent through the stirring speed and the ventilation quantity.

Preferably, the preparation method of the recombinant chicken interferon alpha product expressed by the escherichia coli engineering bacteria comprises the following steps:

(1) fermentation of strains: the engineering bacteria are activated and then inoculated in a fermentation medium according to the inoculation amount of 5-10 percent, fermented at the temperature of 36.8-37.2 ℃, the pH value is controlled to be 6.8-7.2, and the dissolved oxygen is controlled to be more than or equal to 30 percent through the stirring speed and the ventilation quantity;

(2) induction: culturing to bacterial concentration OD_600nmWhen 40 min, IPTG (isopropyl-. beta. -D-thiogalactoside) was added to a final concentration of 0.5-l.0mmol/L to induce interferonExpressing, inducing for 4-6 hr, ending fermentation, and centrifuging to collect wet thallus;

(3) and (3) breaking and cracking thalli: adding the wet bacteria obtained in the step (2) into a culture medium according to a mass-to-volume ratio of 1: (9-11) adding a lysis buffer solution, mixing, performing ultrasonic treatment and centrifugation, and collecting precipitates to obtain a recombinant chicken interferon alpha protein inclusion body;

(4) denaturation and dissolution of inclusion bodies: mixing and dissolving the inclusion body with 10-20 times of volume of denatured dissolving solution, centrifuging, collecting the denatured solution, and removing precipitate to obtain crude recombinant chicken interferon alpha denatured solution; the denatured dissolving solution comprises the following components: 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT, and the balance water;

(5) performing chromatography and purification on an inclusion body: after nickel-sepharose FF chromatographic columns are balanced by using a binding buffer solution, loading the crude recombinant chicken interferon alpha denatured solution at the flow rate of 50-100cm/h, and after loading, removing impure proteins and eluting target proteins by using the binding buffer solution and an elution buffer solution respectively to obtain the purified recombinant chicken interferon alpha denatured solution; the binding buffer consists of: 6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mm imidazole, balance water, pH 7.5;

(6) renaturation of inclusion body proteins: dropping the purified recombinant chicken interferon alpha denatured liquid into the renaturation liquid according to the volume ratio of the recombinant chicken interferon alpha denatured liquid to the renaturation liquid of 1 (14-16), and continuously stirring for renaturation for at least 48h to obtain recombinant chicken interferon alpha renaturation stock solution; the biological activity, namely the specific activity (the ratio of the biological activity to the protein) of the recombinant chicken interferon alpha stock solution is as high as 1.05 multiplied by 10⁹IU/mg;

(7) the recombinant chicken interferon alpha renaturation stock solution is mixed with a protective agent, and is added with water for injection to be diluted and then is subpackaged to obtain a finished product of the recombinant chicken interferon alpha.

The invention provides a production process for thallus crushing, inclusion body denaturation, purification and renaturation, which is suitable for industrial production, and the product purity is more than 95.0%.

Preferably, in the step (1), after culturing for 2.5-3.5h, the feed liquid starts to be supplemented by the rebounding dissolved oxygen, the feed speed is 800mL-1600mL/h, and the feed volume is 5L; the feed liquid comprises the following components in percentage by weight (g/L): glucose: 200, peptone: 10, yeast powder: 10 and the balance of water.

Preferably, in the step (1), the strain activation step is: marking the engineering bacteria on an LB culture dish, culturing overnight at a constant temperature of 37 ℃, and taking the engineering bacteria as first-grade seeds after single bacteria grow out;

streaking the first-stage seeds, inoculating an LB solid culture medium plate, culturing at 36.8-37.2 ℃ for 18-24 hours, transferring a single colony to an LB liquid culture medium, and performing shake culture at 36.8-37.2 ℃ and 200r/min until the thallus concentration OD is obtained_600nmWhen the volume ratio of the bacterial liquid to the culture medium is 3.0, then the ratio of the volume ratio of the bacterial liquid to the volume of the culture medium is 1: inoculating LB liquid culture medium to 10, shaking culturing at 36.8-37.2 deg.C at 200r/min until the thallus concentration of the fermentation liquid reaches 0D600 value of 3.0, and obtaining seed liquid.

Preferably, in the step (2), the centrifugation conditions are as follows: centrifuging at 12000-14000r/min for 15min to 20min at 4 ℃, collecting wet thalli, adding a PBS solution into the wet thalli, mixing according to the mass-volume ratio of 1:10, centrifuging at 12000-14000r/min for 15min to 20min, and collecting the wet thalli; the PBS solution has the composition of NaCl 8.0g/L, KCl 0.2.2 g/L, Na₂HPO₄ 1.44g/L、KH₂PO₄0.24g/L, and the balance of water.

Preferably, in the step (3), the ultrasonic conditions are as follows: carrying out intermittent ultrasonic disruption on bacteria at 2400w power, carrying out ultrasonic treatment for 5s, stopping ultrasonic treatment for 20s, and carrying out ultrasonic treatment for the total time: and (5) 35 min.

Preferably, in the step (3), the lysis buffer consists of: tris 0.05M, EDTA 5mM, NaCl0.1369M, and the balance of water, pH8.5.

Preferably, in the step (3), the centrifugation condition is 12000-14000r/min for 20 min.

Preferably, in the step (4), the centrifugation conditions are 12000-14000r/min and 15-20 minutes.

Preferably, in the step (6), the titration conditions of the renaturation solution are as follows: titration was carried out at 4 ℃ and at a rate of 4-6 mL/min.

Preferably, in the step (7), the protein protectant consists of: 60-80g/L of glucose; preparing Tween 803-5 g/L and water for injection in balance, sterilizing at 118 deg.C for 15min, and cooling at 2-8 deg.C.

Preferably, in the step (7), the recombinant chicken interferon alpha renaturation stock solution and the protective agent are mixed according to a volume ratio of 20: 1, adding water for injection to dilute until the final concentration of the recombinant chicken interferon alpha stock solution is one fifth of the original concentration, fully stirring, and filtering by using a sterilizing filter membrane with the pore diameter of 0.22 mu m to obtain a semi-finished product.

Preferably, in the step (7), the semi-finished product after being subjected to filter sterilization is subpackaged within 24 hours. The subpackaging is carried out according with the product batch and subpackaging regulation of the biological products for animals, and the sealing is carried out under the aseptic condition. The determination of the biological activity of the recombinant chicken interferon alpha in the finished product reaches: 1.0X 10⁸IU/mL-1.1×10⁸IU/mL。

The invention provides a protein protective agent of a product and a proportion thereof.

The recombinant chicken interferon alpha of the organism can well prevent and treat the damage of virus to cells, and the specific activity of the stock solution is not lower than 1.0 multiplied by 10 measured by a cytopathic inhibition method⁹IU/mg, the biological activity of the product is not less than 1.0 multiplied by 10⁸IU/mL. The product has good stability, and the biological activity of the product is not less than 1.0 × 10 after the product is stored at 4 deg.C for 2 years⁸IU/mL。

In the step (5), the aim of balancing the binding buffer solution is to keep the conditions consistent in the protein purification process and avoid the loss of protein due to non-uniform treatment conditions. After the recombinant chicken interferon alpha is loaded, the column is filtered by a binding buffer solution so as to remove the hybrid protein.

In the step (6), compared with the prior art that the inclusion body protein is subjected to denaturation and renaturation and then subjected to affinity chromatography, the inclusion body protein is subjected to denaturation treatment and then subjected to affinity chromatography and renaturation treatment, so that the inclusion body protein is small in treatment amount and convenient to process in a large amount in the purification process.

In the step (6), the addition of arginine and GSSG in the renaturation solution has a significant effect on the renaturation of the protein. The addition of small arginine molecules promotes the correct folding of the protein, and GSSG promotes the correct formation of disulfide bonds, thereby improving the biological activity of the renaturation solution.

The invention also aims to provide a recombinant chicken interferon alpha product expressed by the escherichia coli engineering bacteria prepared by the preparation method.

The invention also aims to provide application of the recombinant chicken interferon alpha product in preparing recombinant protein for preventing avian influenza virus infection.

Preferably, the avian influenza virus is a virus of subtype H9N 2.

Has the advantages that:

point of invention corresponding to technical effect

The invention prepares recombinant chicken interferon alpha, and the stable biological activity is not less than 1.0 multiplied by 10⁸IU/mL of a product comprising:

(1) fermentation of strains: activating the engineering bacteria, inoculating the engineering bacteria into a fermentation culture medium according to the inoculation amount of 10%, fermenting at 37 +/-0.2 ℃, controlling the pH value to be 6.8-7.2, controlling the dissolved oxygen to be more than or equal to 30% through the stirring speed and the ventilation quantity, and supplementing: after about 3 hours of culture, feeding is started after oxygen-dissolved rebounding, the feeding speed is 800mL-1600mL/h, and the feeding volume is 5L. After the culture is carried out for about 3 hours, the dissolved oxygen is adjusted to be more than or equal to 30 percent by the feeding speed of 800mL-1600mL/h, so that the fed carbon source is not excessive and is kept in a dynamic balance state of carbon source consumption and supplement, and finally high-density fermentation is achieved.

(2) Induction: after about 5 hours of fermentation culture, at the moment, the thalli growth is already carried out in the middle and later stages of fermentation logarithm, at the moment, 0.5-l.0mmol/L IPTG (isopropyl-beta-D-thiogalactoside) is added, the expression of interferon is induced, the fermentation is finished after 4-6 hours of induction, and zymocyte liquid is collected to obtain high-expression thalli.

(3) And (3) breaking and cracking thalli: the cells were resuspended in lysis buffer (Tris 0.05M, EDTA 5mM, NaCl 0.8%, pH8.5) (10:1), and disrupted by intermittent sonication at 2400w power for 5s with sonication and 20s with sonication for the total time: and (5) 35 min. Under the condition, the thalli can be fully crushed, and the crushing rate reaches 100 percent through microscopic examination.

(4) Denaturation and dissolution of inclusion bodies: the inclusion body is dissolved fully by using 10-20 times of a solution with 6M guanidine hydrochloride, 1.5MMEDTA, 30mM Tris and 8mM DTT at the temperature of 4 ℃, so that the subsequent protein renaturation is facilitated.

(5) Performing chromatography and purification on an inclusion body: nickel-sepharose FF column: and (3) selecting a nickel-agarose gel FF column to specifically combine with the purified recombinant chicken alpha interferon through an HIS label carried by the recombinant interferon to obtain a purification effect.

The concentration of imidazole in the elution buffer plays an important role in the elution of protein, the concentration of finally obtained protein and the biological activity. The imidazole and the protein are subjected to competitive adsorption on a chromatographic column, and are mixed together with 6M GU-HCl and 20mM tris-HCl under the preferable concentration, namely the concentration of the imidazole is 500mM, so that the recombinant chicken alpha interferon with higher concentration and higher biological activity is obtained.

(6) Renaturation of inclusion body proteins: the purified recombinant chicken interferon alpha denatured stock solution is dripped into a renaturation solution (arginine 0.5M, 2mM EDTA, 0.8mM GSSG, 50mM Tris, 20% glycerol pH:7.0) at the speed of 4-6mL/min under the environment of 4 ℃, and the ratio of the denatured stock solution to the renaturation solution is 1: 15. And continuously stirring for renaturation for at least 48h to obtain recombinant chicken interferon alpha renaturation stock solution, and its biological activity, i.e. specific activity (biological activity and protein ratio) is up to 3.85X 10⁹IU/mg. The invention discloses a method for promoting correct folding of protein by adding small molecular arginine and promoting correct formation of disulfide bond by GSSG, which finds that arginine and GSSG have the function of synergistically improving protein activity in the protein renaturation process, have the characteristic of remarkably improving the biological activity of renaturation liquid under the conditions of 0.5M arginine and 0.8mM GSSG, ensure that recombinant chicken interferon alpha is subjected to industrialized chromatographic separation, still keep remarkable biological activity in the denaturation and renaturation processes, and the specific activity of stock solution is not lower than 1.0 multiplied by 10 after chromatography⁹IU/mg。

Drawings

FIG. 1: lung, spleen and trachea dissection were tested for different batches of interferon treated avian influenza in example 5 of the invention.

FIG. 2: pathological histological observation (40X) of lungs, spleen and trachea of different batches of interferon injection group chickens in example 5 of the invention.

Detailed Description

Example 1 preparation method of recombinant chicken interferon alpha product expressed by Escherichia coli engineering bacteria

The method comprises the steps of producing recombinant chicken interferon alpha BL21/pET-21a-ChIFN alpha engineering bacteria by fermentation in a 50L fermentation tank, carrying out induced expression and crushing, centrifuging, collecting inclusion body precipitates, washing, roughly purifying, then carrying out denaturation and dissolution by guanidine hydrochloride, purifying by nickel ion column affinity chromatography, then carrying out renaturation by a dilution renaturation method, then carrying out filtration sterilization, preparing to obtain stock solution, adding a protective agent, preparing, and detecting to obtain a finished product after the product is qualified.

1.1 seed liquid propagation

The engineering bacteria are firstly inoculated on an LB solid medium plate, cultured for 24 hours at 37 ℃, single colony is shaken to 200ml LB, shaken in a shaking table at 37 ℃ (200r/min) until OD is 3.0, inoculated on a liquid LB medium according to a ratio of 1:10, shaken and cultured at 37 ℃ until OD value is 3.0, and used for inoculation in a fermentation tank.

1.2 fermentation Process

The method comprises the following specific steps:

and (3) high-pressure sterilization: air filter, sampler, ammonia water feeding bottle, defoaming agent feeding bottle, etc.

In-situ sterilization: adding fermentation liquid culture medium into the tank, connecting with pH electrode and dissolved oxygen electrode, and sterilizing in situ.

Inoculation: inoculating secondary seed bacterial liquid according to 10% of the culture medium amount, and fermenting at 37 ℃. The pH value is controlled to be 7.0, and the dissolved oxygen is controlled to be more than or equal to 30 percent through the stirring speed and the ventilation quantity.

Feeding: after about 3 hours of culture, feeding is started after oxygen-dissolved rebounding, the feeding speed is 800ml-1600ml/h, and the feeding volume is 5L.

Induction: the OD600nm value of the cells was measured by sampling at regular intervals. When the OD600nm value of the bacterial liquid reaches 40 (after about 5h of fermentation culture), adding an inducer to start induction expression. And (3) discharging: after inducing for 5h, ending the fermentation, and collecting zymocyte liquid.

Cleaning a fermentation tank: the fermentation tank, the pH probe, the dissolved oxygen probe and all pipelines are washed by pure water, the pH probe is protected in a protective solution, the dissolved oxygen probe is stored in a corresponding box, all screws are installed after the tank is cleaned, after the last fermentation, the pure water is added into the tank for in-situ sterilization, and the controller is closed after the sterilization.

1.3 Collection of cells

Collecting thallus by centrifugation, cooling the fermentation culture to 20 deg.C, centrifuging at 14000r/min for 15min at 4 deg.C, collecting supernatant, and collecting wet thallus. PBS (NaCl 8.0g/L, KCl 0.2.2 g/L, Na2HPO41.44g/L, KH2PO40.24g/L) resuspends (10:1) the wet cells, centrifuges for 15min at 14000 rpm, intensively stores the supernatant, and collects the wet cells. The wet cells were weighed, frozen at-20 ℃ and the batch number, identity, weight and date were indicated on the containers.

1.4 disruption and lysis of the cells

The cells were resuspended in lysis buffer (Tris 0.05M, EDTA 5mM, NaCl 0.8%, pH8.5) (10:1), and disrupted by intermittent sonication at 2400w power for 5s and 20s, with total sonication time of 35 min. And then placing the suspension at 4 ℃, centrifuging at 14000r/min for 20min, intensively storing and treating supernatant fluid, collecting precipitate to obtain crude recombinant chicken interferon alpha protein inclusion bodies, and weighing.

1.5 washing of recombinant Chicken Interferon alpha Inclusion bodies

(1) The pellet was resuspended and washed with PBST (1:10), centrifuged at 12000rpm for 10min at 4 ℃;

(2) resuspending and washing the precipitate with 2M urea (1:10), and centrifuging at 12000rpm for 10min at 4 deg.C;

(3) the precipitate was washed with 1M NaCl (1:10) by resuspension and centrifuged at 12000rpm for 10min at 4 ℃;

TABLE 1 washing formulations of Inclusion bodies

1.6 denaturation

(1) Resuspending the precipitate with denaturant, and stirring the precipitate with denaturant (1:15) at 4 deg.C until the precipitate gradually dissolves;

(2) the mixture was centrifuged at 12000rpm for 15min at 4 ℃ to collect the supernatant.

TABLE 2 denaturant ratios

Name of reagent	Composition of
		Denaturant (PH7.0)	6M guanidine hydrochloride

1.7 purification

Self-loading Ni-Sepharose FF nickel ion chelating affinity chromatography packing of Beijing Boer Sicace, balancing a nickel ion affinity chromatography column by using 3-5 column volumes of binding buffer solution (6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mM imidazole, pH 7.5), detecting an absorption value at a wavelength of 280nm on line, and starting to load the sample after the absorption value is stable; the crude recombinant chicken interferon alpha denatured solution supernatant was applied to a column at a flow rate of 100cm/h, and then passed through a column with a binding buffer (6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mM imidazole, pH 7.5) to wash away the contaminating proteins not bound to the column until A280 stabilized. The eluted protein was collected with an Elution Buffer (6M GU-HCl, 20mM tris-HCl, 500mM imidazole, pH 7.0).

1.8 renaturation

And (3) dripping the purified recombinant chicken interferon alpha denatured liquid into the renaturation liquid at the temperature of 4 ℃ and at the flow rate of 5ml/min, wherein the ratio of the denatured stock solution to the renaturation liquid is 1: 15. And continuously stirring and renaturing for at least 48h to obtain the recombinant chicken interferon alpha renaturation stock solution. The renaturation liquid comprises the following components: arginine 0.5M, 2mM EDTA, 0.8mM GSSG (oxidized glutathione), 50mM Tris, 20% glycerol, balance water, pH 7.0. (% by mass)

TABLE 3 renaturator formulation

1.9 preparation of semi-finished product:

mixing the recombinant chicken interferon alpha stock solution prepared by the method with a protective agent according to the mass-volume ratio of 20: 1, mixing, adding water for injection to the total volume, diluting until the concentration of the recombinant chicken interferon alpha stock solution is one fifth of the original concentration, fully stirring, filtering with a sterilizing filter membrane with the pore diameter of 0.22 mu m to obtain a semi-finished product, and detecting according to the detection standard of the semi-finished product.

Wherein, the proportion of the protective agent, the recombinant chicken interferon alpha stock solution and the water for injection is shown in the following tables 4 and 5. Weighing glucose and Tween 80, adding water for injection, dissolving, sterilizing at 118 deg.C for 15min, and cooling at 2-8 deg.C.

TABLE 4 protectant composition

2.0 preparation of the finished product

According to the existing batch and subpackage regulations of animal biological products in the pharmacopoeia of the people's republic of China.

Example 2 preparation method of recombinant chicken interferon alpha product expressed by Escherichia coli engineering bacteria

(1) Fermentation of strains: the engineering bacteria are activated and then inoculated in a fermentation medium according to the inoculation amount of 5 percent, fermentation is carried out at 37 ℃, the pH value is controlled to be 7.0, and the dissolved oxygen is controlled to be more than or equal to 30 percent through the stirring speed and the ventilation; after 3.0h of culture, feeding liquid begins to be supplemented due to rebound of dissolved oxygen, wherein the feeding speed is 1200mL/h, and the feeding volume is 5L; the feed liquid comprises the following components in percentage by weight (g/L): glucose: 200, peptone: 10, yeast powder: 10 and the balance of water.

The strain activation step comprises: marking the engineering bacteria on an LB culture dish, culturing overnight at a constant temperature of 37 ℃, and taking the engineering bacteria as first-grade seeds after single bacteria grow out;

streaking the first-stage seeds to inoculate an LB solid culture medium plate, culturing at 37 ℃ for 20 hours, transferring a single colony to an LB liquid culture medium, and performing shake culture at 37 ℃ and 200r/min until the single colony is culturedBody concentration OD_600nmWhen the volume ratio of the bacterial liquid to the culture medium is 3.0, then the ratio of the volume ratio of the bacterial liquid to the volume of the culture medium is 1: inoculating 10 LB liquid culture medium, shaking culturing at 37 deg.C and 200r/min until the thallus concentration of the fermentation liquid reaches 0D600 value 3.0, and obtaining seed liquid.

(2) Induction: culturing to bacterial concentration OD_600nmWhen the concentration is 40, adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.5mmol/L, inducing the expression of interferon, finishing fermentation after inducing for 4 hours, and centrifuging to collect wet thalli;

the centrifugation conditions were: centrifuging at 12000r/min for 15min at 4 deg.C, collecting wet thallus, adding PBS solution into the wet thallus, mixing at mass volume ratio of 1:10, centrifuging at 12000r/min for 15min, and collecting wet thallus; the PBS solution has the composition of NaCl 8.0g/L, KCl 0.2.2 g/L, Na₂HPO₄ 1.44g/L、KH₂PO₄0.24g/L, and the balance of water.

(3) And (3) breaking and cracking thalli: adding the wet bacteria obtained in the step (2) into a culture medium according to a mass-to-volume ratio of 1: 9, adding a lysis buffer solution, mixing, performing ultrasonic treatment and centrifugation, and collecting precipitates to obtain a recombinant chicken interferon alpha protein inclusion body;

the ultrasonic conditions are as follows: carrying out intermittent ultrasonic disruption on bacteria at 2400w power, carrying out ultrasonic treatment for 5s, stopping ultrasonic treatment for 20s, and carrying out ultrasonic treatment for the total time: and (5) 35 min. The lysis buffer consisted of: tris 0.05M, EDTA 5mM, NaCl0.1369M, and the balance of water, pH8.5. Centrifuging at 12000r/min for 20 min.

(4) Denaturation and dissolution of inclusion bodies: mixing and dissolving the inclusion body with 10 times of volume of denatured dissolving solution, centrifuging, collecting the denatured solution, and removing precipitate to obtain crude recombinant chicken interferon alpha denatured solution; the denatured dissolving solution comprises the following components: 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT, and the balance water; the centrifugation conditions were 12000r/min, centrifugation 15 min.

(5) Performing chromatography and purification on an inclusion body: after a nickel-sepharose FF chromatographic column is balanced by using a binding buffer solution, loading the crude recombinant chicken interferon alpha denatured solution at the flow rate of 50cm/h, and after loading, removing impure proteins and eluting target proteins by using the binding buffer solution respectively to obtain a purified recombinant chicken interferon alpha denatured solution; the binding buffer consists of: 6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mm imidazole, balance water, pH 7.5;

the elution buffer composition was: 6M GU-HCl, 20mM tris-HCl, 500mM imidazole, balance water, pH 7.5.

(6) Renaturation of inclusion body proteins: dropping the purified recombinant chicken interferon alpha modified solution into the renaturation solution according to the volume ratio of 1:14 to the renaturation solution, and continuously stirring for renaturation for 48 hours to obtain recombinant chicken interferon alpha renaturation stock solution;

the renaturation liquid comprises the following components: arginine 0.5M, 2mM EDTA, 0.8mM GSSG (oxidized glutathione), 50mM Tris, 20% glycerol, balance water, pH 7.0.

The titration conditions of the renaturation solution are as follows: titration was carried out at 5mL/min at 4 ℃.

(7) The recombinant chicken interferon alpha renaturation stock solution and the protective agent are mixed according to the volume ratio of 20: 1, adding water for injection to dilute until the final concentration of the recombinant chicken interferon alpha stock solution is one fifth of the original concentration, fully stirring, and filtering by using a sterilizing filter membrane with the pore diameter of 0.22 mu m to obtain a semi-finished product.

Wherein, the proportion of the protective agent, the recombinant chicken interferon alpha stock solution and the water for injection is the same as that of the example 1, and the details are shown in the following tables 4 and 5.

Example 3 preparation method of recombinant chicken interferon alpha product expressed by Escherichia coli engineering bacteria

The preparation method of the recombinant chicken interferon alpha product expressed by the escherichia coli engineering bacteria comprises the following steps:

(1) fermentation of strains: the engineering bacteria are activated and then inoculated in a fermentation medium according to the inoculation amount of 8 percent, fermentation is carried out at the temperature of 36.8 ℃, the pH value is controlled to be 7.2, and the dissolved oxygen is controlled to be more than or equal to 30 percent through the stirring speed and the ventilation; after 2.5h of culture, feeding liquid begins to be supplemented due to rebound of dissolved oxygen, the feeding speed is 1500mL/h, and the feeding volume is 5L; the feed liquid comprises the following components in percentage by weight (g/L): glucose: 200, peptone: 10, yeast powder: 10 and the balance of water.

streaking the first-stage seeds to an LB solid medium plate, culturing at 37.2 ℃ for 22 hours, transferring a single colony to an LB liquid medium, and performing shake culture at 37.2 ℃ and 200r/min until the thallus concentration OD_600nmWhen the volume ratio of the bacterial liquid to the culture medium is 3.0, then the ratio of the volume ratio of the bacterial liquid to the volume of the culture medium is 1: inoculating LB liquid culture medium to 10, shaking culturing at 37.2 deg.C and 200r/min until the thallus concentration of the fermentation liquor reaches 0D600 value of 3.0, and obtaining seed liquid.

(2) Induction: culturing to bacterial concentration OD_600nmWhen the concentration is 40, adding IPTG (isopropyl-beta-D-thiogalactoside) until the final concentration is 0.5mmol/L, inducing the expression of interferon, finishing fermentation after inducing for 6 hours, and centrifuging to collect wet thalli;

the centrifugation conditions were: centrifuging at 14000r/min for 20min at 4 ℃, collecting wet thalli, adding PBS solution into the wet thalli, mixing according to the mass-to-volume ratio of 1:10, centrifuging at 12000r/min for 15min, and collecting wet thalli; the PBS solution has the composition of NaCl 8.0g/L, KCl 0.2.2 g/L, Na₂HPO₄ 1.44g/L、KH₂PO₄0.24g/L, and the balance of water.

the ultrasonic conditions are as follows: carrying out intermittent ultrasonic disruption on bacteria at 2400w power, carrying out ultrasonic treatment for 5s, stopping ultrasonic treatment for 20s, and carrying out ultrasonic treatment for the total time: and (5) 35 min. The lysis buffer consisted of: tris 0.05M, EDTA 5mM, NaCl0.1369M, and the balance of water, pH8.5. The centrifugation condition is 12000-14000r/min for 20 min.

(4) Denaturation and dissolution of inclusion bodies: mixing and dissolving the inclusion body with 10-20 times of volume of denatured dissolving solution, centrifuging, collecting the denatured solution, and removing precipitate to obtain crude recombinant chicken interferon alpha denatured solution; the denatured dissolving solution comprises the following components: 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT, and the balance water; the centrifugation conditions were 12000r/min, centrifugation 15 min.

(5) Performing chromatography and purification on an inclusion body: after a nickel-sepharose FF chromatographic column is balanced by using a binding buffer solution, loading the crude recombinant chicken interferon alpha denatured solution at the flow rate of 80cm/h, and after loading, removing impure proteins and eluting target proteins by using the binding buffer solution respectively to obtain a purified recombinant chicken interferon alpha denatured solution; the binding buffer consists of: 6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mm imidazole, balance water, pH 7.5;

(6) Renaturation of inclusion body proteins: dropping the purified recombinant chicken interferon alpha modified solution into the renaturation solution according to the volume ratio of the recombinant chicken interferon alpha modified solution to the renaturation solution of 1:16, and continuously stirring for renaturation for at least 48 hours to obtain recombinant chicken interferon alpha renaturation stock solution;

The titration conditions of the renaturation solution are as follows: titration was carried out at 4 ℃ and at a rate of 4-6 mL/min.

Experimental example 1 Activity assay of recombinant Chicken Interferon alpha

The recombinant chicken interferon alpha renaturation stock solution prepared in the embodiments 1, 2 and 3 after the renaturation step is subjected to biological activity measurement, protein content detection, specific activity measurement and purity measurement.

1. Biological Activity assay

The determination method adopts a ' micro cytopathy inhibition method ', specifically refers to the appendix XC in the three parts of pharmacopoeia of the people's republic of China, utilizes chicken embryo fibroblast/vesicular stomatitis virus (CEF/VSV) as a detection system, and uses a national standard to calibrate as an International Unit (IU). (in the prior art, "recombinant human interferon alpha 1b for injection" and "recombinant human interferon alpha 1b injection" all adopt cytopathic inhibition method to determine biological activity)

Through determination, in the embodiments 1-3 of the invention, the titer of the stock solution of the recombinant chicken interferon alpha product is more than 5.0 multiplied by 10⁸IU/ml (see Table 6 below).

TABLE 6 recombinant chicken interferon alpha renaturation stock titer

2. Protein content detection (determination method reference & lt & ltpharmacopoeia of people's republic of China & gt, three appendix VIB second method)

Through determination, in examples 1-3, the protein content of the recombinant chicken interferon alpha product stock solution is more than 0.45mg/ml (see the following table 7).

TABLE 7 recombinant chicken interferon alpha renaturation stock solution protein content

3. Specific activity

The specific activity is the ratio of biological activity to protein content, and is not less than 1.0 × 10⁹IU/mg (see Table 8 below).

TABLE 8 specific Activity of recombinant Chicken Interferon alpha renaturation stock

4. Determination of purity

By using a reducing SDS-polyacrylamide electrophoresis method (the determination method is referred to as appendix IVC of three parts of pharmacopoeia of the people's republic of China)

The concentration of the separation gel is 15%, the sample loading is not lower than 2 mug (Coomassie brilliant blue R250 staining method), and the purity is not lower than 95% by scanning of a scanner (see Table 9 below)

TABLE 9 protein purity of recombinant chicken interferon alpha renaturation stock

Experimental example 2 determination of titer of recombinant chicken interferon alpha finished product

The recombinant chicken interferon alpha products prepared in the examples 1, 2 and 3 are subjected to titer determination:

using cytopathy inhibition: 3 batches of laboratory products, the potency is not less than 1.0 multiplied by 10⁹IU/vial (10 mL).

See in particular Table 10 below

TABLE 10 determination of potency of recombinant chicken interferon alpha products

Experimental example 3 Effect of imidazole concentration in elution buffer on protein recovery

To illustrate the effect of imidazole on protein recovery in the elution buffer, experiments were therefore divided into 6 groups, each corresponding to a different imidazole concentration in the elution buffer, where:

control group 1: 6M GU-HCl, 20mM tris-HCl, 100mM imidazole, balance water, pH 7.5;

control group 2: 6M GU-HCl, 20mM tris-HCl, 200mM imidazole, balance water, pH 7.5;

control group 3: 6M GU-HCl, 20mM tris-HCl, 300mM imidazole, balance water, pH 7.5;

control group 4: 6M GU-HCl, 20mM tris-HCl, 400mM imidazole, balance water, pH 7.5;

control group 5: 6M GU-HCl, 20mM tris-HCl, 600mM imidazole, balance water, pH 7.5;

experimental groups: 6M GU-HCl, 20mM tris-HCl, 500mM imidazole, balance water, pH 7.5.

The experimental method comprises the following steps:

according to step 1.1 of inventive example 1 seed liquid expansion to step 1.7 purification was carried out, the only difference being that in step 1.7 the imidazole concentration in the elution buffer was different. The method is carried out according to the experimental steps, the recovery rate of the protein after the protein is purified by chromatography is measured,

wherein the method for measuring the protein content adopts a second method of Lowry method referring to appendix VIB in three parts of pharmacopoeia of the people's republic of China

The results are detailed in Table 11 below:

TABLE 11 optimization of imidazole concentration in elution buffer

Imidazole concentration (mM)	Protein recovery (%)
		100	35.5
200	42.6
		300	68.7
400	88.5
		500	92.8
600	92.7

Experimental example 4 Effect of arginine and GSSG in renaturation solution on biological activity of recombinant chicken interferon alpha renaturation solution

To illustrate the effect of arginine in conjunction with GSSG on the biological activity of recombinant chicken interferon α refolding solutions, experiments were therefore divided into 13 groups, corresponding to different arginine concentration and GSSG concentration in the elution buffer, respectively, where:

control group 1: 2mM EDTA, 1.0mM GSSG, 50mM Tris, 20% glycerol, pH 7.0;

control group 2: 0.1M arginine, 2mM EDTA, 1.0mM GSSG, 50mM Tris, 20% glycerol, pH 7.0;

control group 3: 0.2M arginine, 2mM EDTA, 1.0mM GSSG, 50mM Tris, 20% glycerol, pH 7.0;

control group 4: 0.3M arginine, 2mM EDTA, 1.0mM GSSG, 50mM Tris, 20% glycerol, pH 7.0;

control group 5: 0.4M arginine, 2mM EDTA, 1.0mM GSSG, 50mM Tris, 20% glycerol, pH 7.0;

control group 6: 0.6M arginine, 2mM EDTA, 1.0mM GSSG, 50mM Tris, 20% glycerol, pH 7.0;

control group 7: 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

control group 8: 0.2mM GSSG, 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

control group 9: 0.4mM GSSG, 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

control group 10: 0.6mM GSSG, 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

control group 11: 1.0mM GSSG, 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

control group 12: 1.2mM GSSG, 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

experimental group 1: 0.8mM GSSG, 0.5M arginine, 2mM EDTA, 50mM Tris, 20% glycerol, pH 7.0;

the experimental method comprises the following steps:

the experiment is divided into 14 groups, the seed solution is respectively expanded and cultured according to the step 1.1 of the embodiment 1 of the invention until the step 1.8 is carried out for renaturation, the recombinant chicken interferon alpha renaturation stock solution is prepared, the only difference is that in the step 1.8, the compositions of the renaturation solution are different, the experiment is respectively carried out according to the groups, the biological activity of the renaturation solution is measured, and the experimental results are detailed in a table 12.

TABLE 12 optimization experiment of arginine addition in renaturation solution

In the renaturation liquid, 0.5M arginine is used in cooperation with 0.8mM GSSG, so that the renaturation liquid has obvious protein activity in the process of realizing the renaturation of the recombinant chicken protein. The addition of arginine and GSSG at higher doses in control 6 and controls 11 and 12 not only increased the reagent cost but also had less effect on the activity of protein renaturation, and even had a tendency to decrease the activity. Experimental example 5 application effect of group chicken interferon alpha product on treating avian influenza

The recombinant chicken interferon alpha products prepared in the examples 1, 2 and 3 are used for carrying out an application effect experiment for treating avian influenza.

1 materials and methods

1.1 materials

1.1.1 Virus chicken H9N2 subtype avian influenza virus G strain

1.1.2 drugs and reagents

Three batches of recombinant chicken interferon a injection (batch number: finished products of examples 1, 2 and 3, titer is 1X 10⁹IU/vial).

1.1.3 test animals 9-11 days old SPF chick embryos and 7 weeks old SPF chicks were purchased from Experimental animals technology, Inc., Meiliya Viton, Beijing.

1.2 methods

1.2.1 animals were grouped and 7-week-old SPF chickens reared in an isolator were randomly divided into a placebo group, a challenge group, a finished batch interferon group of example 1, a finished batch interferon group of example 2, and a finished batch interferon group of example 3, each group consisting of 15 animals, and were fed and drunk freely.

1.2.2 challenge test H9N2G strain seed virus was diluted with sterile normal saline, challenge dose was 10EID 50/cell, the group was inoculated via the wing vein route, interferon group was administered via intramuscular injection, blank control group was inoculated via intramuscular injection with sterile normal saline of the same volume as control, the interferon groups of the finished product batches of examples 1-3 were each infected with H9N2G for 24H, and the interferon products of examples 1-3 were each diluted at a dose of 1 × 10⁶IU/mouse, administered by intramuscular injection for 1 time/day for 3 consecutive days.

1.2.3 clinical symptoms after infection observation, the chicken flocks were observed daily for clinical manifestations, and morbidity and mortality were recorded in detail, with an observation period of 14 days.

1.2.4 Change from Caesarean analysis dead or dying chicks and observing gross specimen changes in trachea, arsine and lung with 3 chickens per group dissected and killed on day 5 post infection

1.2.5 pathological histological observation and killing 3 chickens in each group on the 5 th day after infection, collecting tissues of trachea, lung and spleen for HE staining, and observing histological change.

1.2.6 tissue Virus titer determination on day 5 after infection with strain H9-G, the virus tissue titer was determined after mixing lung tissues from 3 chickens per cell. Mixing and grinding the mixed tissue with liquid nitrogen, freezing and thawing the tissue suspension for three times, centrifuging for 10min at 8000pm, sucking supernatant, adding 1000uUml of double antibody total final concentration for treatment, diluting with normal saline by 10 times in series, respectively inoculating viruses with various dilution degrees into allantoic cavities of 9-11H-old chick embryos, inoculating 5 chick embryos with each dilution degree, incubating at 37 ℃ for 7H, collecting allantoic , and determining HA titer to be positive if the HA titer is not less than 1: 16. The EID50 of the virus is calculated by a Red-Muench method to obtain the virus titer in lung and liver tissues.

2 results

2.1 clinical observations

On the 4 th day after infection, the chickens in the toxin counteracting group breathe by mouth opening and swing heads, and the autopsy can show mucus in the trachea of the larynx, splenic hemorrhage, clove-like substances in the trachea, and only chest swelling and pulmonary congestion can be seen; example 1 finished batch interferon-injected group chickens had mucus in the trachea and bleeding spots in the lungs; example 2 finished batch interferon injection group chicken, the lung was checked by a dissecting test to see the bleeding spot. On day 5 post-infection, 2 deaths were obtained from the challenge group; the rest chickens have the phenomenon of mental depression, respiratory symptoms and rale. On day 6 post-infection, the challenge group chickens breathed mouth-open only. On day 7 after infection, 1 death in the toxin-attacking group was observed, and the examination revealed mucus in the laryngeal trachea, cheese-like substances in the trachea, and blood stasis in the lung. On day 8 post-infection, the chickens in each group were in a recovered state. All the chickens in the blank control group had good spirit and normal ingestion, and no obvious clinical symptoms were seen.

2.2 Caesarean Change

After the 5 th autopsy after the toxin attack, the lung of the toxin attack group has obvious congestion, and the larynx has mucus and massive bleeding points. The lungs of interferon groups showed varying degrees of congestion and a small amount of mucus at the larynx, with the lungs of the finished interferon group of example 2 having congestion and the lungs of the finished interferon group of example 1 and example 3 having a small amount of congestion as shown in figure 1.

2.3 histopathological examination

Lung tissues of the challenge group were infiltrated with exfoliated epithelial cells and inflammatory cell components in bronchi, lung chamber and respiratory capillaries. Bleeding occurs in the bronchi. The white marrow of the spleen was slightly bleeding. The trachea mucosa lamina propria is thickened, and the mucosa lamina propria and submucosa are infiltrated by a large amount of lymph and inflammatory cells. Example 1 bronchodilation was seen in lung tissue from finished interferon batches, and occasional vesicle formation in mucosal lamina propria. Example 2 lung tissue of finished interferon group showed bronchiectasis and a small amount of lymphocyte infiltration in the mucosa lamina propria. Example 3 the lung tissue of the finished interferon batch had hemorrhage in bronchi and a small amount of lymphocyte infiltration in the mucosa lamina propria. As shown in fig. 2.

The pathological changes of each tissue section were evaluated as shown in Table 13 below

TABLE 13 pathological changes evaluation of H9 subtype AIVG Strain infected Chicken Lung, spleen and trachea

Note: "-" indicates no significant pathological change; "+" indicates slight pathological changes were visible; "+ +" indicates the visible portion of the pathological change;

"+ + + +" indicates that severe pathological changes were visible.

2.4 tissue Virus titre assay

5 days after infection, 3 chickens were killed and dissected out of each group, and lung tissues were mixed and the viral tissue titer was determined, with the results shown in Table 14 below.

TABLE 14 Virus titer assay in Lung tissue of different batches of interferon-injected chickens

3 small knot

After the 3.17-week-old SPF chickens are infected with AIVG strains through intravenous injection, obvious clinical symptoms can be observed in the virus attacking group, and clinical symptoms of each batch of interferon are slight compared with the virus attacking group.

3.2 gross necropsy results show that the lung gross lesions of the finished product of example 1 and the finished product of example 3 were slightly more severe than that of the finished product of example 2.

3.3 histological observation of disease states that the pathological changes of the organs of the interferon group of the finished product and the finished product in the example 1 are slightly less than those of the interferon group of the finished product in the example 2

3.4 lung tissue virus titer determination shows that the lung tissue virus content of the finished interferon group of the finished product batch in example 1, the lung tissue virus content of the finished interferon group of the finished product batch in example 2 and the lung tissue virus content of the chicken of the finished product batch in example 3 are all reduced compared with the virus challenge group, and the lung tissue of the chicken of the three batches of interferon groups cannot detect the virus titer, and has significant difference (P is less than 0.01) compared with the virus challenge group.

3.5 in conclusion, the interferon alpha injection for the three batches of recombinant chicken can relieve clinical symptoms, alleviate pathological injuries and reduce the virus titer of lung tissues after the avian influenza infects the chicken.

Sequence listing

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Claims

1. A preparation method of recombinant chicken interferon alpha product expressed by colon bacillus is characterized in that: the method comprises the following steps:

carrying out fermentation culture on escherichia coli engineering bacteria, adding IPTG (isopropyl-beta-thiogalactoside) into the escherichia coli engineering bacteria, finishing fermentation after inducing for 4-6 hours, centrifuging to obtain thalli, crushing and cracking the thalli to obtain a recombinant chicken interferon alpha protein inclusion body, carrying out denaturation and dissolution on the recombinant chicken interferon alpha protein inclusion body, carrying out chromatography purification by using a nickel-sepharose FF chromatography column, carrying out protein renaturation, mixing with a protective agent, adding water for injection for dilution, and subpackaging to obtain a recombinant chicken interferon alpha product;

the renaturation liquid used in the protein renaturation comprises the following components: arginine 0.5M, 2mM EDTA, 0.8mM GSSG, 50mM Tris, 20% glycerol, balance water, pH 7.0;

the elution buffer used in the chromatographic purification consists of: 6M GU-HCl, 20mM tris-HCl, 500mM imidazole, balance water, pH 7.5.

2. The method for preparing recombinant chicken interferon alpha product expressed by colibacillus of claim 1, which is characterized in that: the fermentation culture steps of the escherichia coli engineering bacteria are as follows: the engineering bacteria of the escherichia coli are activated and then inoculated in a fermentation medium according to the inoculation amount of 5-10%, fermentation is carried out at the temperature of 36.8-37.2 ℃, the pH value is controlled to be 6.8-7.2, and the dissolved oxygen is controlled to be more than or equal to 30% through the stirring speed and the ventilation quantity.

3. The method for preparing recombinant chicken interferon alpha product expressed by colibacillus of claim 1, which is characterized in that: the method comprises the following steps:

(1) the engineering bacteria are activated and then inoculated into a fermentation medium according to the inoculation amount of 5-10 percent, and fermented at the temperature of 36.8-37.2 ℃ and the pH value of 6.8-7.2, and the dissolved oxygen is controlled to be more than or equal to 30 percent;

(2) culturing to bacterial concentration OD_600nmWhen the concentration is 40, adding IPTG (isopropyl-beta-D-thiogalactoside) to the final concentration of 0.5-lmmol/L, inducing for 4-6 hours, ending fermentation, and centrifugally collecting wet thalli;

(3) adding the wet bacteria obtained in the step (2) into a culture medium according to a mass-to-volume ratio of 1: (9-11) adding a lysis buffer solution, mixing, performing ultrasonic treatment and centrifugation, and collecting precipitates to obtain a recombinant chicken interferon alpha protein inclusion body;

(4) mixing and dissolving the recombinant chicken interferon alpha protein inclusion body with 10-20 times volume of denatured dissolving solution, centrifuging, collecting the denatured solution, and removing precipitate to obtain crude recombinant chicken interferon alpha denatured solution; the denatured dissolving solution comprises the following components: 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT, and the balance water;

(5) after nickel-sepharose FF chromatographic columns are balanced by using a binding buffer solution, loading the crude recombinant chicken interferon alpha denatured solution at the flow rate of 50-100cm/h, and after loading, removing impure proteins and eluting target proteins by using the binding buffer solution and an elution buffer solution respectively to obtain the purified recombinant chicken interferon alpha denatured solution; the binding buffer consists of: 6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mm imidazole, balance water, pH 7.5;

(6) dropping the purified recombinant chicken interferon alpha denatured liquid into the renaturation liquid according to the volume ratio of the recombinant chicken interferon alpha denatured liquid to the renaturation liquid of 1 (14-16), and continuously stirring for renaturation for at least 48h to obtain recombinant chicken interferon alpha renaturation stock solution;

(7) the recombinant chicken interferon alpha renaturation stock solution is mixed with a protective agent, and is added with water for injection to be diluted and then is subpackaged to obtain a recombinant chicken interferon alpha product.

4. The method for preparing recombinant chicken interferon alpha product expressed by colibacillus of claim 3, which is characterized in that: in the step (1), after culturing for 2.5-3.5h, the feed liquid is supplemented, the feed speed is 800mL-1600mL/h, and the feed volume is 5L; the feed liquid comprises the following components: the feed liquid comprises the following components in percentage by weight (g/L): glucose: 200, peptone: 10, yeast powder: 10 and the balance of water.

5. The method for preparing recombinant chicken interferon alpha product expressed by colibacillus of claim 3, which is characterized in that: in the step (3), the ultrasonic conditions are as follows: carrying out intermittent ultrasonic disruption on thalli at 2400w power, carrying out ultrasonic treatment for 5s, stopping ultrasonic treatment for 20s, and carrying out ultrasonic treatment for the total time: and (5) 35 min.

6. The method for preparing recombinant chicken interferon alpha product expressed by colibacillus of claim 3, which is characterized in that: in the step (7), the protein protective agent comprises the following components: 60-80g/L of glucose; tween 803-5 g/L, and the balance of water for injection, and sterilizing at 118 deg.C for 15 min.

7. The method for preparing recombinant chicken interferon alpha product expressed by colibacillus of claim 6, which is characterized in that: in the step (7), the recombinant chicken interferon alpha renaturation stock solution and the protective agent are mixed according to the volume ratio of 20: 1, adding water for injection to dilute until the final concentration of the recombinant chicken interferon alpha is one fifth of the original concentration, fully stirring, and filtering by using a sterilizing filter membrane with the pore diameter of 0.22 mu m to obtain a semi-finished product.

8. The recombinant chicken interferon alpha product prepared by the preparation method of any one of claims 1 to 7.

9. Use of the recombinant chicken interferon alpha product of claim 8 for the preparation of recombinant proteins for the prevention of avian influenza virus infection.