CN1416894A - Secreted recombinant human interferon alpha-2a injection liquid - Google Patents
Secreted recombinant human interferon alpha-2a injection liquid Download PDFInfo
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- CN1416894A CN1416894A CN 01133662 CN01133662A CN1416894A CN 1416894 A CN1416894 A CN 1416894A CN 01133662 CN01133662 CN 01133662 CN 01133662 A CN01133662 A CN 01133662A CN 1416894 A CN1416894 A CN 1416894A
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Abstract
The secreted recombinant human interferon alpha-2a injection includes secreted recombinant human interferon alpha-2a strain psIFN/WA3110, glycine, mannitol, glucose, sodium chloride, sodium phosphate buffering liquid of pH 7.4 and benzoic alcohol. The secreted recombinant human interferon srhIFN alpha-2a has the first position amino acid residue being cysteine, while other recombinant human interferon rhIFN alpha-2a has the first position amino acid residue being methionine. The gene cloning primer has the structure of 5' AAA GCATGC GAT CTA CCT CAA ACC CAC 3' IFNgene 5' AAA CTT AGG TCA TTC CTTACT TCT TAA 3', where A is adenine, G is guanine, C is cytosine and T is thymine.
Description
Technical field
The present invention relates to a kind of new forms of interferon---the secreted recombinant human interferon alpha-2 injection, particularly escherichia coli secreted recombinant human interferon alpha-2 injection and production technology comprise structure and the secreted recombinant human interferon alpha-2 process for separating and purifying of secreted recombinant human interferon alpha-2 strain psIFN/WA3110.
Background technology
Interferon (IFN) is a kind of activated protein of human secretory, has antiviral, antitumor and immunoregulatory activity widely.Be divided into four hypotypes such as α, β, γ, ω according to its molecular structure and antigenic difference.Alpha-interferon is further divided into hypotypes such as α 1a, α 2a, α 1b, α 2b according to the difference of its structure again, and its difference shows on indivedual amino acid whose differences.The 23rd amino acids residue as human interferon-alpha-2a is a lysine residue, and the 23rd amino acids residue of α-2b is an arginine residues.The antiviral mechanism of interferon-alpha is by inductive effect albumen active cell antiviral activity, induces the release of desmoenzymes such as 2 '-5 '-oligo-adenylate synthetase and double-stranded RNA protein kinase, causes the degraded of viral RNA, thereby suppresses duplicating of virus.Natural interferon is subjected to the restriction of raw material, and output is few, and the price height is restricted its application.
Genetic engineering interferon (recombinant interferon) has and the identical physiological function of natural interferon, and has the advantage that the source is not restricted, output is big, purity is high, price is much lower.The appearance of recombinant interferon (rhIFN) is given human treatment's viral disease and is treated some tumor disease and brought Gospel, and the interferon overwhelming majority of Xiao Shouing all is a recombinant interferon on the market.Clinical trial in nearly 20 years shows that recombinant human interferon-alpha-2a has clear and definite curative effect in treatment viral disease and some malignant tumor.Especially on the treatment Type B viral hepatitis, curative effect is the choice drug of treatment hepatitis B obviously, definitely.
Recombinant human interferon-alpha-the 2a that uses both at home and abroad is not the interferon of natural structure at present, and its first is methionine (Met), and first of the interferon of natural structure is cysteine (Cys).Human body is about 5% to the probability that Met produces antibody.Because the existence of unnecessary Met makes 5% human body produce antibody to rhIFN, make the interferon life-time service after curative effect weaken, influenced the performance of IFN biological function; Present recombinant interferon purifying process complexity, the response rate is lower.Therefore, be necessary to develop active height, be difficult for producing antibody, the simple recombinant interferon of purifying process.
Still there is not secreting type recombinant interferon listing both at home and abroad now, domestic producer that has and research unit, the recombinant interferon that adopted the yeast expression system secreting, expressing.Because this system has added some glycosyl in the protein excretion maturation process, make the easy antibody that produces at glycosyl of rhIFN of secreting, expressing, can influence the result of use of recombinant interferon, therefore, exploitation and development output height, it is very important that the rhIFN that quality is good, cost is low seems.
The technology contents of invention
The objective of the invention is to develop the expression height, product is active high, is difficult for producing antibody, and purifying process is simple, the secreted recombinant human interferon alpha-2 injection that the response rate is high, the particularly structure of rhIFN α-2a and production technology.
Secreted recombinant human interferon alpha-2 injection of the present invention, comprise secreted recombinant human interferon alpha-2 strain psIFN/WA3110, glycine, mannitol, glucose, sodium chloride, sodium phosphate buffer, pH value 7.4, benzyl alcohol are formed, wherein the first amino acids residue of secreting type recombinant human interferon alpha 2 srhIFN α-2a is a cysteine, and the first amino acids residue of other recombinant human interferon alpha 2 rhIFN α-2a is a methionine.
The strain psIFN/WA3110 construction method of above-mentioned secreted recombinant human interferon alpha-2 injection, at first design one couple of PCR primers 1, be used to clone IFN α-2a gene, while is for the ease of the usefulness of clone and structure secretion expression carrier, primer at 5 ' and 3 ' end has been introduced SphI and EcoRI restriction enzyme site respectively, with the synthetic PCR primer of design, TaqDNApolymerase and suitable salt such as mg
2+, Na
+Under the buffer existence condition, carried out following PCR circular response, 90 ℃ of degeneration 3min, 50 ℃ of annealing 2min, 72 ℃ of extension 10min, after carrying out a circulation like this, carry out following 25 circulations: 90 ℃ of degeneration 1min, 55 ℃ of annealing 2min, 72 ℃ of extension 3min, the interferon double-stranded DNA that template DNA oneself prepares for us, promptly ds-cDNA obtains the IFN α-2a genetic fragment of SpHI and EcoRI restriction enzyme site through pcr amplification; Then according to the requirement of secreted expression carrier cloning site, in 5 ' end and 3 ' end PCR primer, design and imported EcoRI and SphI restriction enzyme site respectively, through pcr amplification, obtained containing the dna fragmentation of alkaline phosphatase promoter PhoA and diphtheria corynebacterium exotoxin A fragment signal peptide DTS gene; Again pcr amplification product IFN α-2a gene and the dna fragmentation that contains the PhoA/DTS signal peptide gene are cut liquefaction with EcoRI and SphI, SphI and EcoRI respectively, carry out three sections mixing with carrier PBR322/EcoR1 again and be connected, transform XL-1 blue competence antibacterial with the EcoRI digestion process.Select recombinant clone, obtained containing the recombiant plasmid psIFN of IFN α-2a/PhoA/DTS signal peptide gene.Recombiant plasmid psIFN is transformed the W3110 bacterium, obtained efficient expression engineering strain psIFN/W3110 through expression screening.
Described strain construction method, the structure of gene clone primer 1 is 5 ' AAA GCA TGC GATCTA CCT CAA ACC CAC, 3 ' IFNgene, 5 ' AAA CTT AGG TCA TTC CTT ACT TCTTAA 3 ', and its code name contains and means A: adenine G: guanine C: cytosine T: thymus pyrimidine.
Its primer sequence that makes up efficient expression vector is as follows:
(1) P-EcoRI (promoter primer):
5’TGT?GAA?TTC?AAC?TTC?ATA?CTT?GGC?3’
(2) CP-SpHI (diphtheria toxin, diphtherotoxin signal peptide structure primer)
5’AAA?GCA?TGC?ATG?GGC?TGA?AGG?TGG?3’
Its code name contains and means A: adenine G: guanine C: cytosine T: thymus pyrimidine.
The production technology of above-mentioned secreted recombinant human interferon alpha-2 injection is: the seed liquor preparation-
Fermentation
Centrifugal
Collect thalline
Cracking
Ammonium sulfate is slightly carried
The CM column chromatography
The isoelectrofocusing chromatography
The sephacryls-100 post
The semi-finished product calibrating
Add protective agent
Dilution
Merge
Aseptic filtration
The finished product calibrating
Finished product packing.
The purposes of above-mentioned secreted recombinant human interferon alpha-2 injection is used for the treatment of chronic hepatitis B and chronic hepatitis C, and its usage is intramuscular injection, the next day once, each dosage is from 1000000-3000000IU, be 3-6 month per course of treatment.
We are separating and are cloning on the basis of IFN α-2a gene, having made up with tetracycline and ampicillin is screening sign secreted expression carrier, made up the engineering bacteria of secreting, expressing Recomvinated Interferon on this basis again, screened and efficiently expressed secreting type recombinant interferon (srhIFN) engineering bacteria, developed the pilot scale production technology; At the higher characteristics of secreting type interferon stability, developed and removed the liquid dosage form of the protectant interferon of human albumin.
This liquid state srhIFN preparation and production technology have following characteristics: 1. protein active is tall and big in 2.5 * 10
8IU/mg; 2. the first amino acids residue of recombiant protein is a cysteine, is difficult for producing antibody; 3. purifying process simple (three step purification) does not use monoclonal antibody to carry out purification, (response rate is up to more than 30%); 4. owing in the end used the isoelectrofocusing chromatography in the purge process, improved resolution, final purity of protein is reached more than 98%; 5. protective agent end user's blood albumin not, the potential danger of virus-free pollution; 6. finished product does not need lyophilizing, has reduced production cost.
The specific embodiment
The prescription of every liquid interferon formulation of (1ml) secreting type can be a prescription 1:
srhIFNα-2a 15ug(3000000IU)
Glycine 15mg
Mannitol 2.5mg
Glucose 8mg
Sodium chloride 10mM
Sodium phosphate buffer (pH7.4) 10mM
Benzyl alcohol 10mg prescription 2:
srhIFNα-2a 10ug(2000000IU)
Glycine 15mg
Mannitol 2.5mg
Glucose 8mg
Sodium chloride 10mM
Sodium phosphate buffer (pH7.4) 10mM
Benzyl alcohol 10mg prescription 3:
srhIFNα-2a 7.5ug(1500000IU)
Glycine 15mg
Mannitol 2.5mg
Glucose 8mg
Sodium chloride 10mM
Sodium phosphate buffer (pH7.4) 10mM
Benzyl alcohol 10mg prescription 4:
srhIFNα-2a 5ug(1000000IU)
Glycine 15mg
Mannitol 2.5mg
Glucose 8mg
Sodium chloride 10mM
Sodium phosphate buffer (pH7.4) 10mM
Benzyl alcohol 10mg
1 be that example is narrated its concrete implementing process now: 1. make up efficient expression engineering strain psIFN/W3110 according to the method in the general introduction to write out a prescription.2. the preparation of seed liquor: take from one of the capable psIFN/W3110 freeze-drying lactobacillus that makes up, be inoculated in the LB flat board that contains tetracycline (5 μ g/ml), 37 ℃ of overnight incubation, were inoculated in the 30mlLB culture medium culturing 12 hours with overnight culture in 1: 20 ratio at next day, take out this culture and carry out restriction enzyme mapping mensuration, antibiotic resistance is checked, Gram, electron microscopic examination, be inoculated in the 500mlLB culture medium shaking table shaken cultivation after qualified 5-6 hour, and made seed liquor.3. fermentation culture inserted seed liquor in the SW culture fluid by inoculative proportion 1: 20,26 hours 30 ℃ of bottom fermentation times.4. fermentation culture finishes to collect thalline with continuous centrifuge, and the thalline of results puts-and 70-80 is ℃ frozen.5. restrain the ratio that adds 6ml in thalline 1 and add 1 * TE (10mlTris/HCl, 1mMEDTA, pH8.8 solution), fully mixing is put 4 ℃ of jolting effect 60min, the cracking thalline, and the centrifugal bacterial sediment fragment that goes, supernatant adds ammonium sulfate in the 350g/L ratio and slightly carries.6. purification is crossed CM-sepharose Fast Flow post with crude extract, collects the 10mMPB buffer solution elution peak of pH7.0, crosses the isoelectrofocusing chromatographic column.The isoelectrofocusing post is collected the peak cross molecular sieve chromatography collection main peak.7. the interferon semi-finished product stock solution behind the column chromatography is added protection liquid, according to the Determination of biological activity result, it is diluted to desired concn with aseptic buffer, with 0.22 μ m filter membrane aseptic filtration, sampling is surveyed and is tired sterility test, pyrogen testing.8. the 5ml ampoule (every 1ml) of the diluent branch being packed into lid inertia plug.4 ℃ of following cold preservations promptly get the injection recombinant human interferon alpha 2.
Claims (6)
1. secreted recombinant human interferon alpha-2 injection, comprise secreted recombinant human interferon alpha-2 strain psIFN/WA3110, glycine, mannitol, glucose, sodium chloride, sodium phosphate buffer, pH value 7.4, benzyl alcohol are formed, wherein the first amino acids residue of secreting type recombinant human interferon alpha 2 srhIFN α-2a is a cysteine, and the first amino acids residue of other recombinant human interferon alpha 2 rhIFN α-2a is a methionine.
2. the strain psIFN/WA3110 construction method of the secreted recombinant human interferon alpha-2 injection of claim 1, at first design one couple of PCR primers 1, be used to clone IFN α-2a gene, while is for the ease of the usefulness of clone and structure secretion expression carrier, primer at 5 ' and 3 ' end has been introduced SphI and EcoRI restriction enzyme site respectively, with the synthetic PCR primer of design, TaqDNA polymerase and suitable salt such as mg
2+, Na
+Under the buffer existence condition, carried out following PCR circular response, 90 ℃ of degeneration 3min, 50 ℃ of annealing 2min, 72 ℃ of extension 10min, after carrying out a circulation like this, carry out following 25 circulations: 90 ℃ of degeneration 1min, 55 ℃ of annealing 2min, 72 ℃ of extension 3min, the interferon double-stranded DNA that template DNA oneself prepares for us, promptly ds-cDNA obtains the IFN α-2a genetic fragment of SphI and EcoRI restriction enzyme site through pcr amplification; Then according to the requirement of secreted expression carrier cloning site, in 5 ' end and 3 ' end PCR primer, design and imported EcoRI and SphI restriction enzyme site respectively, through pcr amplification, obtained containing the dna fragmentation of alkaline phosphatase promoter PhoA and diphtheria corynebacterium exotoxin A fragment signal peptide DTS gene; Again pcr amplification product IFN α-2a gene and the dna fragmentation that contains the PhoA/DTS signal peptide gene are cut liquefaction with EcoRI and SphI, SphI and EcoRI respectively, carry out three sections mixing with carrier PBR322/EcoR1 again and be connected, transform XL-1 blue competence antibacterial with the EcoRI digestion process.Select recombinant clone, obtained containing the recombiant plasmid psIFN of IFN α-2a/PhoA/DTS signal peptide gene.Recombiant plasmid psIFN is transformed the W3110 bacterium, obtained efficient expression engineering strain psIFN/W3110 through expression screening.
3, strain construction method according to claim 2, the structure of gene clone primer 1 is 5 ' AAA GCA TGC GAT CTA CCT CAA ACC CAC, 3 ' IFNgene, 5 ' AAA CTT AGGTCA TTC CTT ACT TCT TAA 3 ', and its code name contains and means A: adenine G: guanine C: cytosine T: thymus pyrimidine.
4. strain construction method according to claim 2, the primer sequence that makes up efficient expression vector is as follows: (1) P-EcoRI (promoter primer): 5 ' TGT GAA TTC AAC TTC ATA CTT GGC 3 ' (2) CP-SphI (diphtheria toxin, diphtherotoxin signal peptide structure primer), 5 ' AAA GCA TGC ATG GGC TGA AGG TGG, 3 ' its code name contains and means A: adenine G: guanine C: cytosine T: thymus pyrimidine.
5. the production technology of secreted recombinant human interferon alpha-2 injection in the right 1: seed liquor preparation
Fermentation
Centrifugal
Collect thalline
Cracking
Ammonium sulfate is slightly carried
The CM column chromatography
The isoelectrofocusing chromatography
The sephacryls-100 post
The semi-finished product calibrating
Add protective agent
Dilution
Merge
Aseptic filtration
The finished product calibrating
Finished product packing.
6. the purposes of secreted recombinant human interferon alpha-2 injection in the right 1 is used for the treatment of chronic hepatitis B and chronic hepatitis C, and its usage is intramuscular injection, the next day once, each dosage is from 1000000-3000000IU, be 3-6 month per course of treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01133662 CN1416894A (en) | 2001-11-09 | 2001-11-09 | Secreted recombinant human interferon alpha-2a injection liquid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01133662 CN1416894A (en) | 2001-11-09 | 2001-11-09 | Secreted recombinant human interferon alpha-2a injection liquid |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344491A (en) * | 2003-12-10 | 2012-02-08 | 米德列斯公司 | Interferon alpha antibodies and their uses |
| CN106983716A (en) * | 2017-04-20 | 2017-07-28 | 长春生物制品研究所有限责任公司 | Recombinant human interferon alpha-2 bolt and preparation method |
-
2001
- 2001-11-09 CN CN 01133662 patent/CN1416894A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344491A (en) * | 2003-12-10 | 2012-02-08 | 米德列斯公司 | Interferon alpha antibodies and their uses |
| US8722870B2 (en) | 2003-12-10 | 2014-05-13 | Medarex, L.L.C. | Nucleic acids encoding interferon alpha antibodies |
| CN102344491B (en) * | 2003-12-10 | 2015-03-11 | 梅达雷克斯有限责任公司 | Interferon alpha antibodies and their uses |
| US9765141B2 (en) | 2003-12-10 | 2017-09-19 | E. R. Squibb & Sons, L.L.C. | Methods for preparing interferon alpha antibodies |
| CN106983716A (en) * | 2017-04-20 | 2017-07-28 | 长春生物制品研究所有限责任公司 | Recombinant human interferon alpha-2 bolt and preparation method |
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