CN105879012A

CN105879012A - Production process of viral nephritis treatment medicine interferon

Info

Publication number: CN105879012A
Application number: CN201410693755.4A
Authority: CN
Inventors: 张爱标
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-11-27
Filing date: 2014-11-27
Publication date: 2016-08-24

Abstract

The invention discloses a production process of viral nephritis treatment medicine interferon. The production process comprises the following steps of (1) target gene obtaining; (2) PCR (Polymerase Chain Reaction) amplification: in a microcentrifuge tube, using target genes being 1 <mu>g/mL obtained in the step (1) as template DNA (Deoxyribonucleic Acid), adding a proper amount of a buffer solution, a mixture of four kinds of dNTPs (Deoxy-Ribonucleoside Triphosphates), an DNA polymerase system being 0.5 to 2.5 U/50<mu>L, a pair of DNA synthesis primers being 0.1 to 0.5 <mu>mol/L, and keeping the system PH being 6.8 to 7.8; (3) genetic recombination; (4) transformation; (5) separation and purification; (6) gene expression; (7) modification; and (8) freeze drying. The interferon obtained by the production process provided by the invention can realize the mass production in a short time; after the modification by polyethylene glycol, the half-life period of the interferon is prolonged; the targeting performance on the nephritis viruses is improved; the antigenicity is low; the human body immune reaction cannot be caused; the medicine effect is good; the immobilized production is realized; the stability is high; and the production process is suitable to be popularized and applied.

Description

A kind of production technology of viral nephritis medicine interferon

Technical field

The present invention relates to biomedicine field, particularly relate to the production technology of a kind of viral nephritis medicine interferon.

Background technology

nullIt is many in invisible that viral nephritis infects sequela，Persistent infection can cause uremia and constitutional renal necrosis，Mortality rate is the highest，For being eventually developed to uremic patient，The only effective treatment is just by renal transplantation，But in the case of kidney transfer operation can not be popularized，Chronic viral nephritis is carried out early treatment be just particularly important，At present，The treatment of viral nephritis there is no specific drug，Mainly use interferon、Ucleosides antiviral agents and all kinds of Chinese patent medicine，It it is i.e. immunostimulant，But the interferon half-life on current market is short，Targeting is not enough，More seriously natural interferon medicine has antigen active，Application No. CN200510069269.6 discloses a kind of gene engineered targeted interferon for hepatitis B and production technology thereof，Gene engineered targeted interferon for hepatitis B uses the dsFv gene building people source anti-HBsAg，It is connected with a-interferon (IFN-a) gene，Technique for gene engineering is used to express hepatitis B targeted interferon (dsFv-IFV fusion protein)，The production technology of gene engineered targeted interferon for hepatitis B，The culture medium made is sent into incubator cultivation after inoculation，Complete the amount reproduction between breeding of the antibacterial after cultivating，Then by separating and collecting antibacterial，And crack isolation of occlusion bodies albumen，Renaturation in folding liquid，The most purified、Concentrate，Last subpackage lyophilizing，Prove after affinitive layer purification that this fusion protein has interferon activity and the reactivity with hepatitis B virus surface antigen，Fusion protein belongs to people source，Avoid the harm that heterologous brings，But this interferon still has antigenicity，Additionally targeting is not enough.

Summary of the invention

For overcoming the deficiency in prior art, it is an object of the present invention to provide the production technology of a kind of viral nephritis medicine interferon.

For realizing the purpose of the present invention, technical scheme is as follows:

The production technology of a kind of viral nephritis medicine interferon, comprises the steps:

(1) obtain genes of interest: obtain effector T cell from viral nephritis in the patient, be 37 DEG C in temperature, under conditions of PH is 7.0, with restricted enzyme, DNA is carried out Partial digestion, obtain controlling the genes of interest of interferon synthesis, standby；

(2) PCR amplification: in microcentrifugal tube, with the 1 μ g/mL genes of interest that obtains in step (1) as template DNA, add appropriate buffer, 4 kinds of dNTP (i.e. dATP, dCTP, dGTP, dTTP) mixture, the archaeal dna polymerase system of 0.5-2.5U/50 μ L, primers of a pair 0.1-0.5 μm ol/L synthetic DNA, system PH is that 6.8-7.8, PCR amplification includes following fundamental reaction step:

1. denaturation: temperature 92 DEG C, reacts 5min, and making double-stranded DNA template dissociate becomes strand；

2. deformation: temperature 92 DEG C, reacts 65s, and making double-stranded DNA template dissociate becomes strand

3. renaturation: temperature 55 DEG C, reacts 45s, and primer combines with the pairing of template DNA single-stranded complementary；

4. extending: temperature 72 DEG C, react 5min, " Template-primer conjugate " is in archaeal dna polymerase effect

Under, with dezyribonucleoside as raw material, with target sequence as template, by base pairing and semiconservative replication principle, synthesize new DNA.

5. the circulation of repetition " degeneration--renaturation--extension " 25 times, obtain the genes of interest of requirement；

(3) gene recombinaton: using plasmid as carrier; use the restriction enzyme cleavage plasmid in step (1) and genes of interest; it is made to produce identical cohesive end; during genes of interest after the amplification that archaeal dna polymerase will obtain in step (2) imports plasmid gene again, obtain recombiant plasmid；

(4) convert: escherichia coli are suspended in the 0.4mol/L calcium chloride solution of 3 DEG C, and add the recombiant plasmid in step (3), about half an hour is placed after mixing, the ofest short duration be warming up to 42 DEG C make antibacterial enter heat shock state, addition eutrophy culture medium, to recover, is allowed to be in competence and actively absorbs exogenous genetic material.

(5) isolated and purified: the escherichia coli that incubation step (4) obtains on beef-protein medium, picking contains the bacterium colony of marker gene, it is inoculated in another culture medium cultivation, is repeated picking incubation, until it reaches purity requirement；

(6) gene expression: be placed in fermentation tank cultivation through isolated and purified engineering bacteria, controlling temperature is 37 DEG C, and PH is 6.8, can extract, obtain interferon from fermentation tank effluent；

(7) modify: in reactor, add molecular weight 20000 straight chain mono methoxy polyethylene glycol, step (6) gained interferon, 50mmol/L phosphate buffer, mix homogeneously, temperature be 25 DEG C, PH be 8.5,200r/min concussion reaction 4h, glacial acetic acid with 30% terminates reaction, product filters through 0.45 μm nylon membrane, deaerate after, flowing is allowed to pass through chromatographic column mutually, the method using linear gradient elution and isocratic elution to combine purifies, and obtains modified interferon；

(8) lyophilizing: by diluted sample to 1 × 10⁶IU/ml, adds 2% sucrose, 3% glycine, 0.5% tween 80, and 0.2% gelatin, as protective agent, in subpackage to cillin bottle, every bottle of 1ml, is put into freeze drying box lyophilizing, obtained product.

Preferably, in described step (2), described archaeal dna polymerase is Taq DNA polymerase.

Preferably, in described step (2), described buffer is the dithiothreitol, DTT of KCl, 5mmol/L of the phosphate buffer of 10-50mmol/L, 50mmol/L, the bovine serum albumin of 100 μ g/mL, the Mg of 0.5mmol/L-2mmol/L²⁺Mixed solution.

Preferably, in described step (8), in described protective agent, each content of material is 2% sucrose, 3% glycine, 0.5% tween 80,0.2% gelatin.

nullBeneficial effect: the present invention is by obtaining genes of interest、PCR expands、Gene recombinaton、Convert、Isolated and purified、Gene expression、Modify、The big step of lyophilizing eight completes the production technology of viral nephritis medicine interferon，Can produce the most in a large number，After polyethyleneglycol modified，Extend the half-life of interferon，Improve nephritis virus targeting，And antigenicity is weak，The immunoreation of human body will not be caused，Good drug efficacy，Immobilization produces，Good stability，Whole one-tenth product process strict temperature control and PH，The interferon good stability obtained，Can effectively carry out mass production，In PCR amplification step，It is fast that round pcr has speed，Highly sensitive feature，Carry out according to base pair complementarity principle，Amplification is accurately，Additionally amplification amount is big，Selected archaeal dna polymerase is Taq DNA polymerase，Taq DNA polymerase heat resistance is good，Ensure DNA normal polymerization under the high temperature conditions，100 μ g/mL bovine serum albumin in buffer、Dithiothreitol, DTT has the effect well stablizing enzymatic activity，Mg²⁺It it is the activator of archaeal dna polymerase, affect activity and the verity of enzyme, when concentration is between 0.5mmol/L-2mmol/L, the activity of enzyme is high, use Polyethylene Glycol that interferon is carried out amination modification, add the molecular weight of interferon, stability is improved, reduce excretion of drug, shield the antigenic determinant on some interferon molecule surfaces, reduce immunogenicity, reduce internal clearance rate, and slow down the hydrolysis of protease, extend the half-life of interferon, step of freeze drying adds 2% sucrose, 3% glycine, 0.5% tween 80, 0.2% gelatin, it is effectively guaranteed the biological activity of interferon 1.4 × 10⁸More than IU/mg, and 4 DEG C, still there is the qualitative energy of good temperature in the environment of 6 months.

Detailed description of the invention

For the technological means making the present invention realize, creation characteristic, reach purpose and be easy to understand with effect, below

In conjunction with detailed description of the invention, the present invention is expanded on further.

Embodiment 1:

The production technology of a kind of viral nephritis medicine interferon of the present embodiment, comprises the steps:

(1) genes of interest is obtained: obtain effector T cell from viral nephritis in the patient, be 37 DEG C in temperature,

Under conditions of PH is 7.0, with restricted enzyme, DNA is carried out Partial digestion, obtain controlling the genes of interest of interferon synthesis, standby；

(2) PCR amplification: in microcentrifugal tube, with the 1 μ g/mL genes of interest that obtains in step (1) as template DNA, add appropriate buffer, 4 kinds of dNTP (i.e. dATP, dCTP, dGTP, dTTP) mixture, the archaeal dna polymerase system of 0.5U/50 μ L, primers of a pair 0.1mol/L synthetic DNA, system PH is 6.8, and PCR expands and includes following fundamental reaction step:

(5) isolated and purified: the escherichia coli that incubation step (4) obtains on beef-protein medium, picking contains the bacterium colony of marker gene, it is inoculated in another culture medium cultivation, is repeated 3 times picking incubation, until it reaches purity requirement；

Wherein, in described step (2), described archaeal dna polymerase is Taq DNA polymerase, crucially, in described step (2), described buffer is the dithiothreitol, DTT of KCl, 5mmol/L of the phosphate buffer of 10mmol/L, 50mmol/L, the bovine serum albumin of 100 μ g/mL, the Mg of 0.5mmol/L²⁺Mixed solution, and, in described step (8), in described protective agent, each content of material is 2% sucrose, 3% glycine, 0.5% tween 80,0.2% gelatin.

Embodiment 2:

Remaining is identical with described embodiment 1, and difference is, in described step (2), described archaeal dna polymerase system is the archaeal dna polymerase system of 2.5U/50 μ L, the primer of 0.5 μm ol/L synthetic DNA, and system PH is 7.8,.

Embodiment 3:

Remaining is identical with described embodiment 1, difference is, in described step (2), described archaeal dna polymerase system is the archaeal dna polymerase system of 2U/50 μ L, the primer of 0.3 μm ol/L synthetic DNA, system PH is 7, and in described step (2), described buffer is the dithiothreitol, DTT of KCl, 5mmol/L of the phosphate buffer of 30mmol/L, 50mmol/L, the bovine serum albumin of 100 μ g/mL, the Mg of 1.5mmol/L²⁺Mixed solution.

In above three embodiment, the DNA polymerase activity detected is 0.8 × 10⁸More than IU/mg, PCR expanding effect is obvious, after Polyethylene Glycol amino chemical modification, strand polyethylene glycol-interferon content and modification rate respectively reach 0.25mg/ml and 36.63%, there is the characteristic of typical polyethylene glycol modified protein, exo-antigen activity remains the 15% of native antigen activity, and the interferon after lyophilizing, after 6 months, biological activity is 1.0 × 10⁸More than IU/mg.

nullUnderstand in reality is applied，By obtaining genes of interest、PCR expands、Gene recombinaton、Convert、Isolated and purified、Gene expression、Modify、The big step of lyophilizing eight completes the production technology of viral nephritis medicine interferon，Can produce the most in a large number，After polyethyleneglycol modified，Extend the half-life of interferon，Improve nephritis virus targeting，And antigenicity is weak，The immunoreation of human body will not be caused，Good drug efficacy，Immobilization produces，Good stability，Whole one-tenth product process strict temperature control and PH，The interferon good stability obtained，Can effectively carry out mass production，In PCR amplification step，It is fast that round pcr has speed，Highly sensitive feature，Carry out according to base pair complementarity principle，Amplification is accurately，Additionally amplification amount is big，Selected archaeal dna polymerase is Taq DNA polymerase，Taq DNA polymerase heat resistance is good，Ensure DNA normal polymerization under the high temperature conditions，100 μ g/mL bovine serum albumin in buffer、Dithiothreitol, DTT has the effect well stablizing enzymatic activity，Mg²⁺It it is the activator of archaeal dna polymerase, affect activity and the verity of enzyme, when concentration is between 0.5mmol/L-2mmol/L, the activity of enzyme is high, use Polyethylene Glycol that interferon is carried out amination modification, add the molecular weight of interferon, stability is improved, reduce excretion of drug, shield the antigenic determinant on some interferon molecule surfaces, reduce immunogenicity, reduce internal clearance rate, and slow down the hydrolysis of protease, extend the half-life of interferon, step of freeze drying adds 2% sucrose, 3% glycine, 0.5% tween 80, 0.2% gelatin, it is effectively guaranteed the biological activity of interferon 1.4 × 10⁸More than IU/mg, and 4 DEG C, still there is the qualitative energy of good temperature in the environment of 6 months.

The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalent flow process conversion utilizing description of the invention content to be made, or directly or indirectly it is used in other relevant technical fields, the most in like manner it is included in the scope of patent protection of the present invention.

Claims

1. the production technology of a viral nephritis medicine interferon, it is characterised in that include walking as follows Rapid:

(1) genes of interest is obtained: obtain effector T cell from viral nephritis in the patient, be 37 DEG C in temperature, Under conditions of PH is 7.0, with restricted enzyme, DNA is carried out Partial digestion, obtain controlling interferon and close The genes of interest become, standby；

(2) PCR amplification: in microcentrifugal tube, with the 1 μ g/mL genes of interest obtained in step (1) For template DNA, add appropriate buffer, 4 kinds of dNTP mixture, DNA of 0.5-2.5U/50 μ L gather Synthase system, the primer of a pair 0.1-0.5 μm ol/L synthetic DNA, system PH is that 6.8-7.8, PCR expand Increase and include following fundamental reaction step:

Under, with dezyribonucleoside as raw material, with target sequence as template, by base pairing and semiconservative replication Principle, synthesizes new DNA.

(3) gene recombinaton: using plasmid as carrier, use the restriction enzyme cleavage in step (1) Plasmid and genes of interest so that it is produce identical cohesive end, then through archaeal dna polymerase by step (2) In genes of interest after the amplification that obtains import in plasmid gene, obtain recombiant plasmid；

(4) convert: escherichia coli are suspended in the 0.4mol/L calcium chloride solution of 3 DEG C, and add step (3) recombiant plasmid in, places after mixing about half an hour, the ofest short duration be warming up to 42 DEG C make antibacterial enter heat Shock state, addition eutrophy culture medium, to recover, is allowed to be in competence and actively absorbs exogenic heredity Material.

It is (5) isolated and purified: the escherichia coli that incubation step (4) obtains on beef-protein medium, Picking contains the bacterium colony of marker gene, is inoculated in another culture medium cultivation, repeats picking incubation, Until reaching purity requirement；

(6) gene expression: be placed in fermentation tank cultivation through isolated and purified engineering bacteria, controlling temperature is 37 DEG C, PH is 6.8, can extract, obtain interferon from fermentation tank effluent；

(7) modify: in reactor, add molecular weight 20000 straight chain mono methoxy polyethylene glycol, step (6) Gained interferon, 50mmol/L phosphate buffer, mix homogeneously, temperature be 25 DEG C, PH be 8.5, 200r/min concussion reaction 4h, the glacial acetic acid with 30% terminates reaction, and product is through 0.45 μm nylon After membrane filtration, degassing, allow flowing mutually by chromatographic column, use linear gradient elution and isocratic elution to combine Method purify, obtain modified interferon；

(8) lyophilizing: by diluted sample to 1 × 10⁶IU/ml, adds 2% sucrose, 3% glycine, 0.5% tween -80,0.2% gelatin, as protective agent, in subpackage to cillin bottle, every bottle of 1ml, puts into freeze drying box lyophilizing, Product.

The production technology of viral nephritis medicine interferon the most according to claim 1, its feature Being, in described step (2), described archaeal dna polymerase is Taq DNA polymerase.

The production technology of viral nephritis medicine interferon the most according to claim 1, its feature Being, in described step (2), described buffer is the phosphate buffer of 10-50mmol/L, 50mmol/L The dithiothreitol, DTT of KCl, 5mmol/L, the bovine serum albumin of 100 μ g/mL, 0.5mmol/L-2mmol/L The mixed solution of Mg2+.

The production technology of viral nephritis medicine interferon the most according to claim 1, its feature Be, in described step (8), in described protective agent each content of material be 2% sucrose, 3% glycine, 0.5% Tween 80,0.2% gelatin.