CN111455006A - Recombinant chicken interferon α product expressed by escherichia coli and preparation method and application thereof - Google Patents
Recombinant chicken interferon α product expressed by escherichia coli and preparation method and application thereof Download PDFInfo
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- CN111455006A CN111455006A CN202010280783.9A CN202010280783A CN111455006A CN 111455006 A CN111455006 A CN 111455006A CN 202010280783 A CN202010280783 A CN 202010280783A CN 111455006 A CN111455006 A CN 111455006A
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- recombinant chicken
- interferon
- escherichia coli
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention discloses a recombinant chicken interferon α product expressed by escherichia coli, a preparation method and application thereof, and belongs to the technical field of biology.A recombinant escherichia coli engineering bacterium is subjected to fermentation culture, IPTG is added into the recombinant escherichia coli engineering bacterium, the fermentation is finished after 4-6 hours of induction, a thallus is obtained by centrifugation, the thallus is crushed and cracked to obtain a chicken interferon α protein inclusion body, the chicken interferon α protein inclusion body is subjected to denaturation and dissolution, chromatographic purification is carried out by utilizing a nickel-agarose gel FF chromatographic column, then protein renaturation is carried out, the mixture is mixed with a protective agent, water for injection is added for dilution, and a finished product of the recombinant chicken interferon α is obtained by subpackaging, so that not only is the high-density fermentation of the thallus realized, but also the purification effect and the biological activity of the chicken interferon α protein are remarkably improved.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant chicken interferon α product expressed by escherichia coli, and a preparation method and application thereof.
Background
Since ISaacs and L indenmann found interferons in 1957, interferons have shown extremely strong antiviral and immunoregulatory activities and broad application prospects.
As the interferon has strong antiviral effect, a plurality of research reports are currently used for treating viral diseases of poultry, and a large number of research results show that the recombinant chicken interferon α has obvious antiviral effect when used for preventing and treating viral diseases such as 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 of a recombinant chicken interferon α product expressed by escherichia coli engineering bacteria and an application effect thereof, belonging to a genetic engineering biological product obtained by a molecular biology method.A newly designed chicken α interferon gene sequence is recombined to a pET21a (+) vector, then is transferred to receptor escherichia coli by a transformation mode, and an escherichia coli expression system is adopted to express an exogenous gene, thereby being beneficial to the commercial production of chicken α interferon genes.
Disclosure of Invention
The invention aims to solve the problem of providing a recombinant chicken interferon α product expressed by escherichia coli engineering bacteria, and a preparation method and application thereof.
The Escherichia coli engineering bacteria take B L21 (DE3) as a host, and the constructed engineering strain is E.coli B L21 (DE3)/pET21a (+) -ChIFN α. the engineering strain and the construction method thereof are derived from the invention patent 'recombinant chicken interferon α and a preparation method thereof' with the application number of 201410755900.7.
The cDNA sequence of the recombinant chicken interferon α expressed by the engineering strain is an optimized gene sequence shown in SEQ ID NO. 1.
In order to solve the problems, the invention provides a preparation method of a recombinant chicken interferon α product expressed by escherichia coli engineering bacteria, which comprises the following steps:
carrying out fermentation culture on an escherichia coli engineering bacterium, adding IPTG (isopropyl-beta-thiogalactoside) into the escherichia coli engineering bacterium, finishing fermentation after inducing for 4-6 hours, centrifuging to obtain a bacterium, crushing and cracking the bacterium to obtain a recombinant chicken interferon α protein inclusion body, carrying out denaturation and dissolution on the recombinant chicken interferon α 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 finished product of the recombinant chicken interferon α;
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 α 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 OD600nmWhen the concentration is 40, adding IPTG (isopropyl- β -D-thiogalactoside) to the final concentration of 0.5-l.0mmol/L, inducing the expression of interferon, finishing fermentation after inducing for 4-6 hours, and centrifuging to collect wet thalli;
(3) crushing and cracking the thalli, namely adding a cracking buffer solution into the wet thalli obtained in the step (2) according to the mass-volume ratio of 1 (9-11), mixing, performing ultrasonic treatment and centrifugation, and collecting precipitates to obtain a recombinant chicken interferon α protein inclusion body;
(4) performing denaturation and dissolution on the inclusion body, namely mixing and dissolving the inclusion body with 10-20 times of volume of denaturation and dissolution solution, centrifuging, collecting the denaturation solution, and removing precipitate to obtain crude recombinant chicken interferon α denaturation solution, wherein the denaturation and dissolution solution comprises 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT and the balance of water;
(5) performing inclusion body chromatography purification, namely, after balancing a nickel-agarose gel FF chromatographic column by using a binding buffer solution, loading the crude recombinant chicken interferon α 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 respectively to obtain the purified recombinant chicken interferon α denatured solution, wherein the binding buffer solution consists of 6M GU-HCl, 20M tris-HCl, 0.5M NaCl, 20mm imidazole and the balance of water, and the pH value is 7.5;
(6) renaturation of inclusion body protein, dripping the purified recombinant chicken interferon α denatured liquid into renaturation liquid according to the volume ratio of 1 (14-16) to the renaturation liquid, continuously stirring and renaturing for at least 48h to obtain recombinant chicken interferon α renaturation stock solution, and the biological activity of the recombinant chicken interferon α stock solution, i.e. the specific activity (the ratio of biological activity to protein) is up to 1.05 × 109IU/mg;
(7) the recombinant chicken interferon α renaturation stock solution is mixed with a protective agent, and is added with water for injection to be diluted, and then a finished product of recombinant chicken interferon α is obtained through subpackaging.
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 2.5-3.5h of culture, the dissolved oxygen rebounds to start to supplement the feed liquid, the feeding speed is 800m L-1600m L/h, and the feeding volume is 5L, the feed liquid comprises (g/L) glucose 200, peptone 10, yeast powder 10, and the balance of water.
Preferably, in the step (1), the strain activation step is that the engineering bacteria are streaked on an L B culture dish, and are cultured overnight at a constant temperature of 37 ℃ until single strains grow out, and then the single strains are used as first-class seeds;
streaking the first-stage seeds, inoculating L B solid culture medium plate, culturing at 36.8-37.2 deg.C for 18-24 hr, transferring single colony to L B liquid culture medium, shake culturing at 36.8-37.2 deg.C and 200r/min to OD600nmAnd (3.0), inoculating L B liquid culture medium according to the volume ratio of the bacterial liquid to the culture medium of 1:10, and performing shake culture at 36.8-37.2 ℃ at 200r/min until the bacterial concentration of the fermentation liquid reaches 0D600 value of 3.0 to obtain the seed liquid.
Preferably, in the step (2), the centrifugation condition is that the wet thallus is centrifuged for 15min-20min at the temperature of 12000-14000r/min under 4 ℃, PBS solution is added into the wet thallus and mixed according to the mass-volume ratio of 1:10, and the wet thallus is centrifuged for 15min-20min at the temperature of 12000-14000r/min, wherein the PBS solution comprises 8.0 g/L of NaCl, 0.2 g/L of KCl and Na2HPO41.44g/L、KH2PO40.24 g/L, and the balance 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 condition of the renaturation solution is that titration is carried out at the speed of 4-6m L/min at the temperature of 4 ℃.
Preferably, in the step (7), the protein protectant comprises glucose 60-80 g/L, tween 803-5 g/L and water for injection in balance, and is sterilized at 118 ℃ for 15min and cooled (2-8 ℃) for later use.
Preferably, in the step (7), the reconstituted chicken interferon α stock solution and the protective agent are mixed according to the volume ratio of 20: 1, and water for injection is added to dilute until the final concentration of the reconstituted chicken interferon α stock solution is one fifth of the original concentration, the mixture is fully stirred, and a semi-finished product is obtained after the filtration by a sterilization filter membrane with the pore size of 0.22 mu m.
Preferably, in the step (7), the semi-finished product after filtration sterilization is packaged within 24 hours, the packaged product meets the requirements of product batch and packaged specification of veterinary biological products, the packaged product is sealed under aseptic condition, and the biological activity of the recombinant chicken interferon α in the finished product is measured to reach 1.0 × 108IU/mL-1.1×108IU/mL。
The invention provides a protein protective agent of a product and a proportion thereof.
The biological recombinant chicken interferon α can well prevent and treat the damage of virus to cells, and the specific activity of the stock solution is not less than 1.0 × 10 measured by a cytopathic inhibition method9IU/mg, the biological activity of the product is not less than 1.0 × 108IU/m L, the product stability is good, and the biological activity of the product is still not less than 1.0 × 10 after the product is stored for 2 years at 4 DEG C8IU/mL。
In the step (5), the aim of the solution balance of 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, and after the recombinant chicken interferon α is loaded, the aim of passing the binding buffer solution through a chromatographic column is 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 α 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 α product in preparing recombinant proteins 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 α, and the obtained recombinant chicken interferon is stable and has biological activity not lower than 1.0 × 108IU/m L product, comprising:
(1) and (2) fermenting the strain, namely activating the engineering bacteria, inoculating the engineering bacteria into a fermentation culture medium according to the inoculation amount of 10%, fermenting at the temperature of 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 amount, feeding, namely after about 3 hours of culture, feeding by rebounding the dissolved oxygen, wherein the feeding speed is 800m L-1600m L/h, and the feeding volume is 5L, after about 3 hours of culture, adjusting the dissolved oxygen to be more than or equal to 30% through the feeding speed of 800m L-1600m L/h, so that the fed carbon source is not excessive, and the dynamic balance state of carbon source consumption and supplement is kept, and finally high-density fermentation is achieved.
(2) Inducing, 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, adding IPTG (isopropyl- β -D-thiogalactoside) with the final concentration of 0.5-l.0 mmol/L to start the expression of the induction interferon, finishing the fermentation after 4-6 hours of induction, and collecting zymocyte liquid 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) And (3) performing chromatography and purification of the inclusion body, namely selecting a nickel-agarose gel FF column, and obtaining the purification effect by specifically combining the nickel-agarose gel FF column with the purified recombinant chicken α interferon through an HIS label carried by the recombinant interferon.
Imidazole and protein are subjected to competitive adsorption on a chromatographic column, and under the preferable concentration, namely the concentration of imidazole is 500mM, the imidazole is mixed with 6M GU-HCl and 20mM tris-HCl to obtain the recombinant chicken α interferon with higher concentration and higher biological activity.
(6) Renaturation of inclusion body protein, namely dripping the purified recombinant chicken interferon α denatured stock solution into the renaturation solution (arginine 0.5M, 2mM EDTA, 0.8mM GSSG, 50mM Tris, 20% glycerol pH:7.0) at the speed of 4-6M L/min at the temperature of 4 ℃, wherein the ratio of the denatured stock solution to the renaturation solution is 1:15, continuously stirring for renaturation for at least 48h to obtain the recombinant chicken interferon α renaturation stock solution, and the biological activity, namely the specific activity (the ratio of the biological activity to the protein) is as high as 3.85 × 109IU/mg, the correct folding of protein is promoted by adding small molecular arginine, and the correct formation of disulfide bond is promoted by GSSG, the invention discovers that arginine and GSSG have the function of synergistically improving the activity of protein in the protein renaturation process, and have the characteristic of obviously improving the biological activity of renaturation liquid under the conditions of 0.5M arginine and 0.8mM GSSG, so that the recombinant chicken interferon α still keeps the obvious biological activity in the processes of denaturation and renaturation in the industrialized chromatographic separation process, and the specific activity of the stock solution is still kept after chromatographyThe performance is not lower than 1.0 × 109IU/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 shows histopathological observations of lung, spleen and trachea of different batches of interferon-injected group chickens in example 5 of the present invention (40 ×).
Detailed Description
Example 1 preparation method of recombinant chicken interferon α product expressed by Escherichia coli engineering bacteria
The method comprises the steps of producing recombinant chicken interferon α B L21/pET-21 a-ChIFN α strain by fermentation in a 50L fermentation tank, carrying out induced expression and crushing, centrifugally collecting inclusion body precipitate, washing and roughly purifying the inclusion body, carrying out denaturation and dissolution by guanidine hydrochloride, purifying by nickel ion column affinity chromatography, then carrying out renaturation on the inclusion body precipitate by a dilution renaturation method, preparing a stock solution after filtering and sterilizing, adding a protective agent, preparing, and detecting to obtain a finished product after the finished product is qualified.
1.1 seed liquid propagation
The engineering bacteria are firstly inoculated on L B solid medium flat plates and cultured for 24 hours at 37 ℃, single colony is shaken to 200ml L B, shaking table shaking at 37 ℃ is carried out (200r/min) until OD is 3.0, liquid L B culture medium is inoculated according to the proportion of 1:10, and shaking culture is carried out at 37 ℃ until OD value is 3.0 for inoculation of 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, namely feeding after about 3 hours of culture, wherein the feeding is started after oxygen dissolved rebounds, 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 thalli by centrifugation, cooling the fermentation culture to 20 ℃, centrifuging for 15min at 14000r/min under the condition of 4 ℃, collecting supernatant fluid for centralized storage treatment, collecting wet thalli, resuspending (10:1) the wet thalli by PBS (NaCl 8.0 g/L, KCl 0.2 g/L, Na2HPO41.44g/L and KH2PO40.24g/L), centrifuging for 15min at 14000r, performing centralized storage treatment on the supernatant fluid, collecting the wet thalli, weighing the wet thalli, freezing and storing at-20 ℃, and marking batch numbers, tank types, weights and dates on containers.
1.4 disruption and lysis of the cells
The thalli is resuspended by lysis buffer (Tris 0.05M, EDTA 5mM, NaCl 0.8%, pH8.5) (10:1), the bacteria is broken by intermittent ultrasound with 2400w power for 5s and 20s, the total time of ultrasound is 35min, the suspension is placed at 4 ℃, centrifuged at 14000r/min for 20min, the supernatant is stored and treated in a centralized way, the precipitate is collected, and crude recombinant chicken interferon α protein inclusion bodies are obtained and weighed.
1.5 washing of inclusion bodies of recombinant Chicken interferon α
(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-assembly was carried out using Ni-Sepharose FF nickel ion chelating affinity chromatography packing from Beijing BoercoC by equilibrating the nickel ion affinity chromatography column with 3-5 column volumes of binding Buffer (6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mM imidazole, pH7.5) and detecting the 280nm wavelength absorbance on-line and starting the loading after it stabilized, loading the crude recombinant chicken interferon α supernatant onto the column at a flow rate of 100cm/h, then loading the column with binding Buffer (6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mM imidazole, pH7.5), washing off the contaminating proteins that did not bind to the column until A280 stabilized, and then collecting the eluted protein with Elution Buffer (6M GU-HCl, 20mM tris-HCl, 500mM imidazole, pH 7.0).
1.8 renaturation
Dropping the purified recombinant chicken interferon α denatured liquid into renaturation liquid at the flow rate of 5ml/min at the temperature of 4 ℃, wherein the proportion of the denatured stock solution and the renaturation liquid is 1:15, continuously stirring and renaturing for at least 48h to obtain the recombinant chicken interferon α renaturation stock solution, and the renaturation liquid comprises 0.5M arginine, 2mM EDTA, 0.8mM GSSG (oxidized glutathione), 50mM Tris, 20% glycerol and the balance of water, and the pH value is 7.0 (%) (% by mass)
TABLE 3 renaturator formulation
1.9 preparation of semi-finished product:
mixing the stock solution of the recombinant chicken interferon α prepared by the above steps with a protective agent according to the mass-volume ratio of 20: 1, adding water for injection to the total volume, diluting until the concentration of the stock solution of the recombinant chicken interferon α is one fifth of the original concentration, fully stirring, filtering by 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 α stock solution and the water for injection is detailed in the following tables 4 and 5. glucose and Tween 80 are weighed and weighed, and are added with the water for injection for dissolution, sterilized for 15min at 118 ℃, and cooled (2-8 ℃) for standby.
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 α product expressed by Escherichia coli engineering bacteria
(1) And (2) fermenting the strain, namely inoculating the activated engineering bacteria into a fermentation culture medium according to the inoculation amount of 5%, fermenting at 37 ℃, controlling the pH value to be 7.0, controlling the dissolved oxygen to be more than or equal to 30% through the stirring speed and the ventilation amount, after culturing for 3.0 hours, feeding liquid is started to be supplemented after the dissolved oxygen rebounds, wherein the feeding speed is 1200m L/h, and the feeding volume is 5L, and the feed liquid comprises (g/L) glucose 200, peptone 10, yeast powder 10 and the balance of water.
The strain activation step comprises marking the engineering strain on L B culture dish, culturing at 37 deg.C overnight, and taking the strain as first-stage seed after single strain grows out;
the first-stage seeds are streaked and inoculated into L B solid medium plates, after the first-stage seeds are cultured for 20 hours at 37 ℃, single colonies are transferred to L B liquid medium, and shake culture is carried out at 37 ℃ and 200r/min until the thallus concentration OD is reached600nmAnd (3.0), inoculating L B liquid culture medium according to the volume ratio of the bacterial liquid to the culture medium of 1:10, and performing shake culture at 37 ℃ at 200r/min until the thallus concentration of the fermentation liquor reaches 0D600 value of 3.0 to obtain the seed liquid.
(2) Induction: culturing to bacterial concentration OD600nmWhen the concentration is 40, adding IPTG (isopropyl- β -D-thiogalactoside) to the final concentration of 0.5 mmol/L, inducing the expression of interferon, finishing fermentation after inducing for 4 hours, and collecting wet thalli by centrifugation;
centrifuging at 4 deg.C at 12000r/min for 15min, 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, wherein the PBS solution comprises NaCl 8.0 g/L, KCl 0.2 g/L, Na2HPO41.44g/L、KH2PO40.24 g/L, and the balance water.
(3) Crushing and cracking the thallus, namely adding a cracking buffer solution into the wet thallus obtained in the step (2) according to the mass-to-volume ratio of 1: 9, mixing, performing ultrasonic treatment and centrifugation, and collecting precipitate to obtain a recombinant chicken interferon α 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) Performing denaturation and dissolution on the inclusion body, namely mixing and dissolving the inclusion body with 10 times of volume of denaturation and dissolution solution, centrifuging, collecting the denaturation solution, removing the precipitate to obtain crude recombinant chicken interferon α denaturation solution, wherein the denaturation and dissolution solution comprises 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT and the balance of water, and the centrifugation condition is 12000r/min and the centrifugation is carried out for 15 minutes.
(5) Performing inclusion body chromatography purification, namely, after balancing a nickel-agarose gel FF chromatographic column by using a binding buffer solution, loading the crude recombinant chicken interferon α 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 the purified recombinant chicken interferon α denatured solution, wherein the binding buffer solution consists of 6M GU-HCl, 20mMtris-HCl, 0.5M NaCl, 20mm imidazole and the balance of water, and the pH value is 7.5;
the elution buffer composition was: 6M GU-HCl, 20mM tris-HCl, 500mM imidazole, balance water, pH 7.5.
(6) Renaturation of the inclusion body protein, namely dripping the purified recombinant chicken interferon α denatured liquid into the renaturation liquid according to the volume ratio of the denatured liquid to the renaturation liquid of 1:14, and continuously stirring for renaturation for 48h to obtain recombinant chicken interferon α 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 renaturation solution was titrated at 4 ℃ at 5m L/min.
(7) Mixing the recombined chicken interferon α renaturation stock solution and the protective agent according to the volume ratio of 20: 1, adding injection water for dilution until the final concentration of the recombined chicken interferon α stock solution is one fifth of the original concentration, fully stirring, and filtering by a sterilization filter membrane with the aperture of 0.22 mu m to obtain a semi-finished product.
Wherein, the proportion of the protective agent, the recombinant chicken interferon α stock solution and the water for injection is the same as that in example 1, and the details are shown in the following tables 4 and 5.
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%.
Example 3 preparation method of recombinant chicken interferon α product expressed by Escherichia coli engineering bacteria
The preparation method of the recombinant chicken interferon α product expressed by the escherichia coli engineering bacteria comprises the following steps:
(1) and (2) fermenting the strain, namely activating the engineering bacteria, inoculating the engineering bacteria into a fermentation culture medium according to 8% of inoculation amount, fermenting at the temperature of 36.8 ℃, controlling the pH value to be 7.2, controlling the dissolved oxygen to be more than or equal to 30% through stirring speed and ventilation, after culturing for 2.5 hours, feeding liquid is started to be supplemented due to the rebound of the dissolved oxygen, wherein the feeding speed is 1500m L/h, and the feeding volume is 5L, and the feed liquid comprises (g/L) glucose 200, peptone 10, yeast powder 10, and the balance of water.
The strain activation step comprises marking the engineering strain on L B culture dish, culturing at 37 deg.C overnight, and taking the strain as first-stage seed after single strain grows out;
streaking the primary seeds, inoculating L B solid culture medium plates, culturing at 37.2 ℃ for 22 hours, transferring single colonies to L B liquid culture medium, and performing shake culture at 37.2 ℃ and 200r/min until the thallus concentration OD600nmWhen the volume ratio of the bacterial liquid to the culture medium is 3.0, inoculating L B liquid culture medium according to the volume ratio of 1:10, and performing shake culture at 37.2 ℃ at 200r/min until the bacterial concentration of the fermentation liquid reaches 0D600 value of 3.0 to obtain the seed liquid.
(2) Induction: culturing to bacterial concentration OD600nmWhen the concentration is 40, adding IPTG (isopropyl- β -D-thiogalactoside) to the final concentration of 0.5 mmol/L, inducing the expression of interferon, finishing fermentation after 6 hours of induction, and collecting wet thalli by centrifugation;
centrifuging at 14000r/min for 20min 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, wherein the PBS solution comprises NaCl 8.0 g/L, KCl 0.2 g/L, and Na2HPO41.44g/L、KH2PO40.24 g/L, and the balance water.
(3) Crushing and cracking the thallus, namely adding a cracking buffer solution into the wet thallus obtained in the step (2) according to the mass-to-volume ratio of 1: 9, mixing, performing ultrasonic treatment and centrifugation, and collecting precipitate to obtain a recombinant chicken interferon α 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. The centrifugation condition is 12000-14000r/min for 20 min.
(4) Performing denaturation and dissolution on the inclusion body, namely mixing and dissolving the inclusion body with 10-20 times of volume of denaturation and dissolution solution, centrifuging, collecting the denaturation solution, and removing precipitate to obtain crude recombinant chicken interferon α denaturation solution, wherein the denaturation and dissolution solution comprises 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT and the balance of water, and the centrifugation condition is 12000r/min and the centrifugation time is 15 minutes.
(5) Performing inclusion body chromatography purification, namely, after balancing a nickel-agarose gel FF chromatographic column by using a binding buffer solution, loading the crude recombinant chicken interferon α 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 the purified recombinant chicken interferon α denatured solution, wherein the binding buffer solution consists of 6M GU-HCl, 20mMtris-HCl, 0.5M NaCl, 20mm imidazole and the balance of water, and the pH value is 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 protein, namely dripping the purified recombinant chicken interferon α denatured liquid into the renaturation liquid according to the volume ratio of the denatured liquid to the renaturation liquid of 1:16, and continuously stirring for renaturation for at least 48h to obtain recombinant chicken interferon α 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 condition of the renaturation solution is that titration is carried out at the speed of 4-6m L/min at the temperature of 4 ℃.
(7) Mixing the recombined chicken interferon α renaturation stock solution and the protective agent according to the volume ratio of 20: 1, adding injection water for dilution until the final concentration of the recombined chicken interferon α stock solution is one fifth of the original concentration, fully stirring, and filtering by a sterilization filter membrane with the aperture of 0.22 mu m to obtain a semi-finished product.
Wherein, the proportion of the protective agent, the recombinant chicken interferon α stock solution and the water for injection is the same as that in example 1, and the details are shown in the following tables 4 and 5.
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%.
Experimental example 1 Activity assay of recombinant Chicken Interferon α
The recombinant chicken interferon α renaturation stock solution prepared in the example 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 an appendix XC in 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 national standard products to calibrate as an International Unit (IU) (in the prior art, the ' recombinant human interferon α 1b for injection ' and the ' recombinant human interferon α 1b injection ' all adopt a cytopathy 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 α product is more than 5.0 × 108IU/ml (see Table 6 below).
TABLE 6 reconstituted chicken interferon α titer of reconstituted stock solution
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 original liquid of the recombinant chicken interferon α product is greater than 0.45mg/ml (see the following table 7).
TABLE 7 recombinant chicken interferon α renaturation stock solution protein content
3. Specific activity
The specific activity is the ratio of biological activity to protein content, and the specific activity is not less than 1.0 × 109IU/mg (see Table 8 below).
TABLE 8 specific Activity of recombinant Chicken Interferon α renaturation stock solution
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 α renaturation stock solution
Experimental example 2 determination of titer of recombinant chicken interferon α finished product
The recombinant chicken interferon α products prepared in examples 1, 2 and 3 were subjected to titer determination:
the cell lesion inhibiting method is adopted, and the titer of 3 batches of laboratory products is not less than 1.0 × 109IU/bottle (10m L).
See in particular Table 10 below
TABLE 10 determination of the potency of the recombinant Chicken Interferon α Final product
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 L owry method and refers to appendix VIB of three parts of pharmacopoeia of 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 α renaturation solution
To illustrate the effect of arginine in conjunction with GSSG in the renaturation solution on the biological activity of recombinant chicken interferon α renaturation solution, 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 α 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 solution, 0.5M arginine is utilized to cooperate with 0.8mM GSSG, so that the protein renaturation of the recombinant chicken is realized, and the protein activity is obvious, while the contrast group 6 and the contrast groups 11 and 12 are added with arginine and GSSG in larger doses, so that the reagent cost is continuously improved, the influence on the activity of the protein renaturation is small, and even the activity is reduced, in the experimental example 5, the application effect of the chicken interferon α product on treating avian influenza is realized
The recombinant chicken interferon α products prepared in 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 1 × 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 sterilized normal saline, the challenge dose was 10EID 50/single, the group was inoculated via the wing vein route, interferon group was administered via intramuscular injection, the blank control group was inoculated via intramuscular injection with sterilized normal saline of the same volume as the control, the interferon groups of the finished product batches of examples 1-3 were infected with H9N2G for 24H, and the interferon products of examples 1-3 were each administered 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 three batches of recombinant chicken interferon α injection can relieve clinical symptoms, alleviate pathological injuries and reduce lung tissue virus titer after avian influenza infects chickens.
Sequence listing
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Claims (10)
1. A preparation method of a recombinant chicken interferon α product expressed by escherichia coli is characterized by comprising the following steps:
carrying out fermentation culture on an escherichia coli engineering bacterium, adding IPTG (isopropyl-beta-thiogalactoside) into the escherichia coli engineering bacterium, finishing fermentation after inducing for 4-6 hours, centrifuging to obtain a bacterium, crushing and cracking the bacterium to obtain a recombinant chicken interferon α protein inclusion body, carrying out denaturation and dissolution on the recombinant chicken interferon α 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 to dilute, and subpackaging to obtain a recombinant chicken interferon α 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.
2. The process for preparing recombinant chicken interferon α expressed by Escherichia coli according to claim 1, wherein the elution buffer used in the chromatographic purification comprises 6M GU-HCl, 20mM tris-HCl, 500mM imidazole, and the balance water, pH 7.5.
3. The method for preparing recombinant chicken interferon α product expressed by Escherichia coli according to claim 1, wherein the fermentation culture of Escherichia coli engineering bacteria comprises activating the Escherichia coli engineering bacteria, inoculating 5% -10% of the bacteria in a fermentation medium, fermenting at 36.8-37.2 deg.C, controlling pH to 6.8-7.2, and controlling dissolved oxygen to be greater than or equal to 30% by stirring speed and ventilation.
4. The preparation method of the recombinant chicken interferon α product expressed by Escherichia coli according to claim 2, which 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 OD600nmWhen the concentration is 40, adding IPTG to the final concentration of 0.5-lmmol/L, inducing for 4-6 hours, ending fermentation, and centrifugally collecting wet thalli;
(3) adding a lysis buffer solution into the wet thalli obtained in the step (2) according to the mass-volume ratio of 1 (9-11), mixing, carrying out ultrasonic treatment and centrifugation, and collecting precipitates to obtain a recombinant chicken interferon α protein inclusion body;
(4) mixing and dissolving the recombinant chicken interferon α 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 α denatured solution, wherein the denatured dissolving solution comprises 6M guanidine hydrochloride, 1.5mM EDTA, 30mM Tris, 8mM DTT and the balance of water;
(5) after nickel-sepharose FF chromatographic columns are balanced by using a binding buffer solution, loading the crude recombinant chicken interferon α 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 respectively to obtain purified recombinant chicken interferon α denatured solution, wherein the binding buffer solution comprises 6M GU-HCl, 20mM tris-HCl, 0.5M NaCl, 20mm imidazole and the balance of water, and the pH value is 7.5;
(6) dropping the purified recombinant chicken interferon α denatured liquid into the renaturation liquid according to the volume ratio of the denatured liquid to the renaturation liquid of 1 (14-16), and continuously stirring for renaturation for at least 48h to obtain recombinant chicken interferon α renaturation stock solution;
(7) the recombinant chicken interferon α renaturation stock solution is mixed with a protective agent, and is added with water for injection to be diluted, and then the product of the recombinant chicken interferon α is obtained through subpackage.
5. The method for preparing recombinant chicken interferon α expressed by Escherichia coli according to claim 4, wherein in step (1), after culturing for 2.5-3.5h, a feed solution is fed at a feed rate of 800m L-1600m L/h and a feed volume of 5L, wherein the feed solution comprises (g/L) glucose 200, peptone 10, yeast powder 10, and the balance water.
6. The method for preparing recombinant chicken interferon α product expressed by Escherichia coli according to claim 4, wherein in step (3), the ultrasonication is performed at 2400w power for intermittent ultrasonication of thallus for 5s, and the ultrasonication is stopped for 20s, and the total ultrasonication time is 35 min.
7. The method for preparing the recombinant chicken interferon α product expressed by Escherichia coli according to claim 4, wherein in step (7), the protein protectant comprises glucose 60-80 g/L, Tween 803-5 g/L, and water for injection in balance, and the protein protectant is sterilized at 118 ℃ for 15 min.
8. The method for preparing recombinant chicken interferon α product expressed by Escherichia coli according to claim 7, wherein in step (7), the recombinant chicken interferon α renaturation stock solution and the protective agent are mixed according to the volume ratio of 20: 1, and the mixture is diluted with water for injection until the final concentration of the recombinant chicken interferon α is one fifth of the original concentration, and the mixture is fully stirred and filtered by a sterilization filter membrane with the pore size of 0.22 μm to obtain a semi-finished product.
9. The recombinant chicken interferon α product prepared by the preparation method of any one of claims 1-8.
10. Use of the recombinant chicken interferon α product of claim 9, for the preparation of recombinant proteins for the prevention of avian influenza virus infection.
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