CN1990862A - Production method of recombined human alpha1-thymus peptide and preparation thereof - Google Patents
Production method of recombined human alpha1-thymus peptide and preparation thereof Download PDFInfo
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- CN1990862A CN1990862A CN 200510112134 CN200510112134A CN1990862A CN 1990862 A CN1990862 A CN 1990862A CN 200510112134 CN200510112134 CN 200510112134 CN 200510112134 A CN200510112134 A CN 200510112134A CN 1990862 A CN1990862 A CN 1990862A
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Abstract
The invention discloses a method for preparing human alpha 1- thymosin and the human alpha 1- thymosin agent produced with said method. The method comprises: constructing gene engineering bacteria for human alpha 1- thymosin, liquid culturing gene engineering bacteria and fermentating, and puring the recombined human alpha 1- thymosin. The method is characterized by no limit to raw material source, low cost, feasibility for large- scale production, and the product can be used as injection agent for preventing and curing and assistant curing for disease caused by low immune function.
Description
Technical field
The present invention relates to biological pharmacy technical field, be specifically related to the production method and the preparation of recombinant human alpha 1-Zadaxin.
Background technology
α 1-Zadaxin (α 1-thymosin, T α 1) be that Goldstein equals to find in thymic tissue first in 1977, mainly be present in thymocyte, T-lymphocyte and the thymic tissue in vivo, at organs such as liver, kidney, the heart, lung, spleens distribution is arranged also, its precursor α 1-Zadaxin former (prothymosin) of being made up of 111 amino acid adds down through enzymolysis and produces in vivo.Sophisticated people α 1-Zadaxin is made up of 28 amino acid, molecular weight 3.108KD, and iso-electric point 4.2, its sequence is:
Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-ILe-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn。
The normal concentration of people α 1-Zadaxin in human serum is approximately 540-670pg/mL, its major physiological function is for regulating the immunocompetence of body, comprise and stimulate multipotential stem cell to be divided into thymocyte, promote the differentiation and the maturation of T-cell, strengthen the function of T cell, and make CD3+ and the isocellular quantity rising of CD4+ etc.As a kind of wide spectrum immunomodulator, α 1-Zadaxin clinical application comprises and is used for the treatment of B-mode, hepatitis C; As immune ancillary drug; Be used for tumour and treating AIDS etc.This stranger α 1-Zadaxin also with the regulation and control of cell cycle, angiogenic growth, cell migration, the penetrativitys of tissue repair and sperm etc. are relevant.
The Zadaxin extensive market is an example with the treatment hepatitis B only, and the whole world has 2,000,000,000 people once to infect hepatitis B virus approximately, wherein has 3.5 hundred million patients to be converted into the hepatitis B patient.China is one of hepatitis B high incidence country, estimates to have approximately 1.2 hundred million hepatitis B patients.Method at the present most pronounced effects of treatment of hepatitis B is exactly to replenish cytokine, regulate autoimmunity, α 1-Zadaxin separately or the acting in conjunction of associating other drug be one of prefered method of treatment viral hepatitis, will produce the market sale near 1,000,000,000 yuan every year.
The Zadaxin of domestic production at present is to extract from animal thymus mostly, because extract is a mixture, effective constituent concentration is low, and curative effect is not ideal enough, and contains animal proteinum in the mixture, may cause allergic reaction during injection.And, make quality control more difficult because the source is stable inadequately.American-European countries such as the present U.S. have forbidden clinical use animal-origin Zadaxin medicine.
In recent years state stranger α 1-Zadaxin medicine adopts the chemical synthesis process preparation more, the advantage of chemosynthesis is that product purity is with active higher, but traditional polypeptide synthesis method generally can only synthesize the following polypeptide of 10 amino acid, for 28 amino acid whose α 1-Zadaxin, synthetic difficulty is very big, output is lower, is difficult to satisfy market-oriented needs.For example every content of Zadaxin medicine of commodity " Zadaxin " by name is 1.6mg, and price is more than 800 yuan, and be more than 50,000 yuan a course of treatment, costs an arm and a leg, and common patient is difficult to bear.
Summary of the invention
At the deficiency that prior art exists, one of purpose of the present invention be to provide a kind of can be on a large scale, the method for low cost production people α 1-Zadaxin.
Another object of the present invention is to provide the people α 1-Thymopeptide of producing by this method.
Specifically, the present invention is by engineered means, and the clone obtains people α 1-thymosin gene and is connected to expression vector, makes up the recombination engineering bacteria, by high density fermentation and purifying, prepares highly purified recombinant human alpha 1-Zadaxin polypeptide protein.
The technical scheme that realizes objects of the present invention is as follows:
A kind of production method of people α 1-Zadaxin, this method in turn includes the following steps:
(1) structure of people α 1-thymosin gene engineering bacteria: α 1-thymosin gene is synthetic or increase from the human cDNA library by the PCR method and to obtain by chemical process, its synthetic gene comprises SD sequence-purification tag-protease cutting site or chemical cracking point-people α 1-thymosin gene-terminated base sequence, pass through endonuclease digestion then, people α 1-thymosin gene is cloned in the expression vector, and expression vector changes the host bacterium over to and obtains genetic engineering bacterium:
(2) genetic engineering bacterium that structure is finished carries out liquid culture and fermentation, induces or temperature-induced by IPTG, expresses a large amount of soluble recombining people α 1-Zadaxin; Described liquid culture and the employed substratum of fermentation are a kind of in LB substratum and the M9 substratum or the mixture of the two, and the proportioning of the two is: LB: M9=1: 0.1-1: 10;
(3) purifying of recombinant human alpha 1-Zadaxin: the recombinant human alpha 1-Zadaxin of fermentative production obtains fusion rotein through bacterial cell disruption, centrifugal, concentrated, chromatography means, by proteolytic cleavage, chemical cracking mode, obtain the recombinant human alpha 1-Zadaxin polypeptide of higher degree in conjunction with affinity chromatography and desalination;
Described step (1) is specifically: a, by the aminoacid sequence of people α 1-Zadaxin, the following dna segment of chemosynthesis: 5 '-CCT CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATATAC ATA TGT CTG GAT CAG GTC ATC ATC ATC ATC ATC ATT CTT CTG GTACCG ATG ACG ACG ACA AGA GCG ATG CCG CCG TGG ATA CCA GCA GCGAAA TTA CCA CCA AAG ATC TGA AAG AAA AAA AAG AAG TGG TGG AAGAAG CCG AAA ACT AAG ACT AGT GAA TTC AC-3 '; B, with this segment endonuclease digestion, gel reclaims, and makes up PET series expression vector or pGEM plasmid, the transformed competence colibacillus intestinal bacteria, the picking positive colony carries out the sequencing of recombinant plasmid; C, the expression vector that sequence is correct transform the host bacterium, obtain genetic engineering bacterium;
Wherein the purification tag described in (1) step is His-tag or GST, proteolytic enzyme is enteropeptidase, zymoplasm or Xa factor, described chemical cracking reagent is cyanogen bromide, employed expression vector comprises PET series, pGEM plasmid, described host bacterium is intestinal bacteria, comprises DH5 α, BL21 (DE3) or BLR (DE3).
Get thalline in the described step (3) and add in proportion in the broken bacterium damping fluid of imidazoles-NaCl-PB, carry out the broken bacterium of high-pressure homogenization, broken bacterium working pressure is 70Mpa～80Mpa, centrifugal collection supernatant liquor.The supernatant liquor of centrifugal collection is packed in the metallic nickel ions chelating affinity column, and imidazoles-the NaCl-PB balance is steady to baseline with damping fluid, and the pH value is 8.0, imidazoles-NaCl-PB wash-out collector α 1-thymic peptide fusion protein as one main peak; Re-use Phenyl Sepharose and carry out hydrophobic chromatography, collect and penetrate the peak, SDS-PAGE detects; Use the G-25 desalination, collect α 1-thymic peptide fusion protein as one main peak; 10 times of dilutions are dissolved among the PB that contains CaCl2, and the pH value is 7.0, add enteropeptidase and carry out enzymolysis; Use Source 30RPC medium behind the enzymolysis, carry out reversed phase chromatography on AKTA explorer, gradient elution is collected main peak, and removes acetonitrile through the G-25 desalination and promptly obtain highly purified recombinant human alpha 1-Zadaxin.
Described bacterial cell disruption method comprises N,O-Diacetylmuramidase cracking, chemical cracking, ultrasonication, high-pressure homogenization, and described chromatography method comprises the combination of affinity chromatography, hydrophobic chromatography, reversed phase chromatography, ion chromatography or several method.
The recombinant human alpha 1-Zadaxin polypeptide stoste that the purity that obtains is higher, the damping fluid and the protective material that under aseptic condition, add the water for injection configuration as requested, dilute, and carry out freeze-drying later on, preparation injection recombinant human alpha 1-Sterile Lyophilized Preparation Thymosin at sealed packaging;
Contain recombinant human alpha 1-Zadaxin 0.5-30mg in described every injection; Described protective material comprise N.F,USP MANNITOL, glycerine, glycine, sucrose, lactose, glucose wherein one or more.
The recombinant human alpha 1-Zadaxin of the present invention's preparation, use separately or unite use with other medicines, in order to treat or to prevent diseases such as viral influenza, severe acute respiratory syndrome (SARS), hepatitis, immunologic hypofunction, malignant tumour, perhaps as adjuvant drug, in order to the recovery of immunologic function behind the chemotherapy of tumors.
People α 1-Zadaxin preparation method of the present invention has can guarantee the quality product consistence, not limited by raw material sources, and cost is lower, can scale operation etc. many advantages, the product of being produced can be used as injection formulations, is used for prevention, treatment and the assisting therapy of diseases such as immunologic hypofunction.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention.
Fig. 1 is the building process figure of expression plasmid pET-32a-T α 1;
Fig. 2 is a process flow sheet of the present invention;
The purifying process schema of Fig. 3 recombinant human alpha 1-Zadaxin.
Embodiment
Below in conjunction with specific embodiment and accompanying drawing 1-Fig. 3, be described in further detail the present invention and how implement.Embodiment is intended to set forth for example embodiments of the present invention, rather than limits the present invention in any form.Those skilled in the art are according to enlightenment of the present invention, and the various changes of doing in conjunction with the general knowledge of this area are all in the scope that patent application right of the present invention requires.
Used plasmid, thalline etc. in specification sheets and following examples, and the experimental technique of unreceipted actual conditions can be undertaken by the normal condition that those skilled in the art grasped, or undertaken by the condition that goods supplier is advised.
The construction of genetic engineering of high expression level people α 1-Zadaxin:
1, people α 1-thymosin gene total length base sequence obtains: by the aminoacid sequence of people α 1-Zadaxin, and synthetic following dna segment:
5’-CCT?CTA?GAA?ATA?ATT?TTG?TTT?AAC?TTT?AAG?AAG?GAG?ATA?TACATA?TGT?CTG?GAT?CAG?GTC?ATC?ATC?ATC?ATC?ATC?ATT?CTT?CTG?GTA?CCGATG?ACG?ACG?ACA?AGA?GCG?ATG?CCG?CCG?TGG?ATA?CCA?GCA?GCG?AAATTA?CCA?CCA?AAG?ATC?TGA?AAG?AAA?AAA?AAG?AAG?TGG?TGG?AAG?AAGCCG?AAA?ACT?AAG?ACT?AGT?GAA?TTC?AC-3’
This fragment comprises SD sequence, purification tag His-tag, enteropeptidase restriction enzyme site, total length people α 1-thymosin gene and terminator codon successively.
2, this fragment is used the XbaI/EoRI double digestion in 37 ℃ of water-baths, gel reclaims, and the big fragment that reclaims with the XbaI/EoRI double digestion of pET-32a (+) is connected construction of expression vector pET-32a-T α 1 (as shown in Figure 1); The competent cell of transformed into escherichia coli DH5 α preparation; The picking positive colony is identified with enzyme cutting method, PCR method and sequencing;
3, pET-32a-T α 1 carrier that Sequence Identification is correct transforms the host bacterium, and amplification cultivation uses IPTG to induce, and collects thalline, ultrasonication, and centrifugal and collection supernatant liquor, the SDS-PAFE electrophoresis showed has a large amount of people α 1-thymic peptide fusion protein as one to express after inducing.
Embodiment 2
The genetic engineering bacterium fermentation
1, the genetic engineering bacterium that structure is finished is got to be inoculated in the 100ml LB substratum and (is contained the 100ug/ml penbritin), 37 ℃ of shake-flask culture 12-14h; Draw bacterium liquid according to 10% inoculum size and join in the 500ml LB substratum, 37 ℃, 230r/min shake-flask culture 6h finishes the seed liquor preparation;
2, in 30 liters of fermentor tanks (Switzerland Bioengineering company), seed liquor is equipped with in the fermentor tank of 12L M9+LB substratum by the adding of 5% inoculum size, 37 ℃, 300r/min cultivates, air flow is 10L/min, control pH value is about 7.0, dissolved oxygen control is more than 30%, be cultured to OD600 and be about at 12 o'clock, begin to add supplemented medium, be about at 18 o'clock, add IPTG and induce in OD600, to final concentration be 0.8mmol/L, control dissolved oxygen curve and pH substantially steadily but slightly descend receive bacterium after inducing 4h, finish fermentation.
Embodiment 3
Recombinant human alpha 1-Zadaxin purifying (referring to accompanying drawing 2)
1, collects thalline, 6000～7000g Continuous Flow or centrifugal in batches, the every batch of centrifugation time 8～10 minutes; Precipitate thalline and suspend with broken bacterium damping fluid [30mmol/L imidazoles-200mmol/L NaCl-20mmol/L PB (pH8.0)], carry out the broken bacterium of high-pressure homogenization, the working pressure that adopts when breaking bacterium is 70Mpa～80Mpa, the centrifugal impurity of removing after the broken bacterium of microscopy is finished;
2, preparation metallic nickel ions chelating affinity column is got Chelating Sepharose FF 180mL (Amersham Biosciences company) dress post, hangs Ni with 3 column volumes of 50mmol/L NiSO4 solution washing
2+The above-mentioned centrifuged supernatant supernatant liquor of ultrafiltration and concentration (or by) is gone up sample, steady with damping fluid [30mmol/L imidazoles-200mmol/L NaCl-20mmol/L PB (pH8.0)] balance to baseline; 60mmol/L imidazoles-200mmol/L NaCl-20mmol/L PB (pH8.0) drip washing foreign protein is to the baseline balance, 100mmol/L imidazoles-200mmol/L NaCl-20mmol/L PB (pH8.0) wash-out collector α 1-thymic peptide fusion protein as one main peak; Re-use Phenyl Sepharose (Amersham Biosciences company) 20ml and carry out hydrophobic chromatography, collect and penetrate the peak, SDS-PAGE detects;
3, use G-25 50mL desalination, collect α 1-thymic peptide fusion protein as one main peak; 10 times of dilutions are dissolved among the 20mmol/L PB (pH7.0) that contains 1mmol/L CaCl2, add enteropeptidase and carry out enzymolysis; Metallic nickel ions chelating affinity column on the solution behind the enzymolysis, 80mmol/L imidazoles-100mmol/L NaCl-20mmol/L PB (pH8.0) wash-out obtains α 1-Zadaxin polypeptide; Use Source 30RPC 20mL medium, carry out reversed phase chromatography on AKTA explorer, (moving phase is A liquid to gradient elution: 20mmol/L KH
2PO
4(pH5.9), B liquid: acetonitrile; B liquid is from 0-30%) collect main peak, and remove the recombinant human alpha 1-Zadaxin that acetonitrile promptly obtains purity＞95% through the G-25 desalination.
The key technical indexes and the detection method of the 1-Zadaxin that the present invention obtains are as follows:
1, people α 1-Zadaxin molecular weight determination:
Adopt trishydroxymethyl glycine-SDS-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) method to measure, preparation 16%T, the separation gel of 6%C and 6%T, the concentrated glue of 3%C (90mm * 70mm * 0.75mm).About 1.5 hours of the 50V of elder generation constant voltage, after sample entered separation gel, voltage was raised to 70V about 2 hours, through 0.2% Coomassie brilliant blue R-250 dyeing, compared with reference substance and molecular weight standard.Both apparent molecular weights are identical, but because of containing more negative charge amino acid, are not inconsistent with theoretical value (3108).
2, people α 1-Zadaxin isoelectric point determination:
Isoelectrofocusing-polyacrylamide gel electrophoresis adopts Pharmacia LKB company instrument, and glassine paper is prepared 7.5% glue and done the gel diaphragm as supporting film.The gel diaphragm is layered on 4 ℃ the flat board, puts the yin, yang electrode strip that is soaked with electrode solution respectively, regulating voltage 2000V, electric current 10mA, power 10W carries out prerunning 10min, regulating voltage 2000V behind the application of sample, electric current 15mA, power 15W, electrophoresis 80min cuts off the electricity supply, finish poly-glue, observe band position, primary area.Sample P I value is 3.79, and is more smaller than reference substance, is to lack acetylizad result in the N section.
3, people α 1-Zadaxin purity testing
Detecting with the high pressure liquid chromatography (HPLC) method, is weighting agent (Hypersil BDS250mm * 4.6mm, 5 μ m) with octadecylsilane chemically bonded silica; With phosphate buffered saline buffer (potassium primary phosphate 9.5 grams, be dissolved in the 1600mL water, with 1mol/L NaOH adjust pH to 5.7, thin up is to 2000m L) be mobile phase A, the second eyeball is a Mobile phase B, flow velocity is 1.0m L/min, press among the 0-20min Mobile phase B from the gradient elution of 0-30%, the detection wavelength is 210mm, and sample size is 20 μ L, and recording sample purity is 98.3%.
4, people α 1-Zadaxin biological activity determination:
According to literature method (" bioactivity research of synthesizing thymosins α 1 ", the medicine biotechnology, 1998,5 (2): 103), biological activity is so that the rate that increases of the function of its E acceptor of thymus gland T cellular-restoring after taking off the E acceptor is a representation unit, all under the mass action of 100 μ g/mL, it is 24.39% that sample sets increases rate, is better than control group 19.17% at reference substance and sample.
SEQUENCE?LISTING
＜110〉Huaxin Advanced Biotechnical Co., Ltd., Shanghai
＜120〉recombinant human alpha 1-Zadaxin production method and preparation
<140>CurrentAppNumber：200510112134.3
<141>CurrentFilingDate：2005-12-28
<160>1
<170>PatentIn?version?3.3
<210>1
<211>206
<212>DNA
<213>Human
<400>1
cctctagaaa?taattttgtt?taactttaag?aaggagatat?acatatgtct?ggatcaggtc 60
atcatcatca?tcatcattct?tctggtaccg?atgacgacga?caagagcgat?gccgccgtgg 120
ataccagcag?cgaaattacc?accaaagatc?tgaaagaaaa?aaaagaagtg?gtggaagaag 180
ccgaaaacta?agactagtga?attcac 206
Claims (7)
1, a kind of production method of people α 1-Zadaxin, this method in turn includes the following steps:
(1) structure of people α 1-thymosin gene engineering bacteria: α 1-thymosin gene is synthetic or increase from the human cDNA library by the PCR method and to obtain by chemical process, its synthetic gene comprises SD sequence-purification tag-protease cutting site or chemical cracking point-people α 1-thymosin gene-terminated base sequence, pass through endonuclease digestion then, people α 1-thymosin gene is cloned in the expression vector, and expression vector changes the host bacterium over to and obtains genetic engineering bacterium;
(2) genetic engineering bacterium that structure is finished carries out liquid culture and fermentation, induces or temperature-induced by IPTG, expresses a large amount of soluble recombining people α 1-Zadaxin; Described liquid culture and the employed substratum of fermentation are a kind of in LB substratum and the M9 substratum or the mixture of the two, and the proportioning of the two is: LB: M9=1: 0.1-1: 10;
(3) purifying of recombinant human alpha 1-Zadaxin: the recombinant human alpha 1-Zadaxin of fermentative production obtains fusion rotein through bacterial cell disruption, centrifugal, concentrated, chromatography means, by proteolytic cleavage, chemical cracking mode, obtain the recombinant human alpha 1-Zadaxin polypeptide of higher degree in conjunction with affinity chromatography and desalination.
2, the production method of people α 1-Zadaxin according to claim 1, it is characterized in that: described step (1) specifically: a, by the aminoacid sequence of people α 1-Zadaxin, the following dna segment of chemosynthesis: 5 '-CCT CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATATAC ATA TGT CTG GAT CAG GTC ATC ATC ATC ATC ATC ATT CTTCTG GTA CCG ATG ACG ACG ACA AGA GCG ATG CCG CCG TGG ATACCA GCA GCG AAA TTA CCA CCA AAG ATC TGA AAG AAA AAA AAGAAG TGG TGG AAG AAG CCG AAA ACT AAG ACT AGT GAA TTC AC-3 '; B, with this segment endonuclease digestion, gel reclaims, and makes up PET series expression vector or pGEM plasmid, the transformed competence colibacillus intestinal bacteria, the picking positive colony carries out the sequencing of recombinant plasmid; C, the expression vector that sequence is correct transform the host bacterium, obtain genetic engineering bacterium.
3, according to the production method of claim 1 or the 2 any described people α of claim 1-Zadaxin, it is characterized in that: wherein the purification tag described in (1) step is His-tag or GST, proteolytic enzyme is enteropeptidase, zymoplasm or Xa factor, described chemical cracking reagent is cyanogen bromide, employed expression vector comprises PET series, pGEM plasmid, described host bacterium is intestinal bacteria, comprises DH5 α, BL21 (DE3) or BLR (DE3).
4, according to the production method of claim 1 or the 2 any described people α of claim 1-Zadaxin, it is characterized in that: get thalline in the described step (3) and add in proportion in the broken bacterium damping fluid of imidazoles-NaCl-PB, carry out the broken bacterium of high-pressure homogenization, broken bacterium working pressure is 70Mpa～80Mpa, centrifugal collection supernatant liquor.
5, the production method of people α 1-Zadaxin according to claim 4, it is characterized in that: the supernatant liquor of centrifugal collection is packed in the metallic nickel ions chelating affinity column, steady with damping fluid imidazoles-NaCl-PB balance to baseline, the pH value is 8.0, imidazoles-NaCl-PB wash-out collector α 1-thymic peptide fusion protein as one main peak; Re-use Phenyl Sepharose and carry out hydrophobic chromatography, collect and penetrate the peak, SDS-PAGE detects; Use the G-25 desalination, collect α 1-thymic peptide fusion protein as one main peak; 10 times of dilutions are dissolved among the PB that contains CaCl2, and the pH value is 7.0, add enteropeptidase and carry out enzymolysis; Use Source 30RPC medium behind the enzymolysis, carry out reversed phase chromatography on AKTA explorer, gradient elution is collected main peak, and removes acetonitrile through the G-25 desalination and promptly obtain highly purified recombinant human alpha 1-Zadaxin.
6, the high purity recombinant human alpha 1-Zadaxin stoste that claim 1-5 is obtained; the damping fluid and the protective material that under aseptic condition, add the water for injection configuration; dilute; and carry out freeze-drying later at sealed packaging; prepare injection recombinant human alpha 1-Sterile Lyophilized Preparation Thymosin, contain recombinant human alpha 1-Zadaxin 0.5-30mg in every injection.
7, recombinant human alpha 1-Sterile Lyophilized Preparation Thymosin according to claim 6 is characterized in that: described protective material comprise N.F,USP MANNITOL, glycerine, glycine, sucrose, lactose, glucose wherein one or more.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191303A (en) * | 2010-11-26 | 2011-09-21 | 扬子江药业集团北京海燕药业有限公司 | Method for expressing and preparing gene recombinant Talpha1 |
| CN102660568A (en) * | 2012-03-28 | 2012-09-12 | 深圳市海王英特龙生物技术股份有限公司 | A method for preparing recombinant thymulin alpha 1 |
| CN105886581A (en) * | 2015-04-03 | 2016-08-24 | 北京诺思兰德生物技术股份有限公司 | High-density fermentation method of recombinant human thymalfasin |
| CN110554081A (en) * | 2019-10-17 | 2019-12-10 | 东莞太力生物工程有限公司 | Isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein |
-
2005
- 2005-12-28 CN CN 200510112134 patent/CN1990862A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191303A (en) * | 2010-11-26 | 2011-09-21 | 扬子江药业集团北京海燕药业有限公司 | Method for expressing and preparing gene recombinant Talpha1 |
| CN102660568A (en) * | 2012-03-28 | 2012-09-12 | 深圳市海王英特龙生物技术股份有限公司 | A method for preparing recombinant thymulin alpha 1 |
| CN102660568B (en) * | 2012-03-28 | 2013-05-08 | 江苏海王生物制药有限公司 | A method for preparing recombinant thymulin alpha 1 |
| CN105886581A (en) * | 2015-04-03 | 2016-08-24 | 北京诺思兰德生物技术股份有限公司 | High-density fermentation method of recombinant human thymalfasin |
| CN110554081A (en) * | 2019-10-17 | 2019-12-10 | 东莞太力生物工程有限公司 | Isoelectric point detection method of isoelectric focusing electrophoresis of recombinant protein |
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