CN1873006A - Method for producing recombined human proinsulin - Google Patents
Method for producing recombined human proinsulin Download PDFInfo
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- CN1873006A CN1873006A CN 200510026290 CN200510026290A CN1873006A CN 1873006 A CN1873006 A CN 1873006A CN 200510026290 CN200510026290 CN 200510026290 CN 200510026290 A CN200510026290 A CN 200510026290A CN 1873006 A CN1873006 A CN 1873006A
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Abstract
This invention provides a method for producing recombinant human proinsulin from N-end leader peptide, and relevant expression vector. The method comprises: (1) fusing yeast alpha-factor leader peptide sequence and the human proinsulin N-end leader peptide, and introducing into a yeast constitutive expression vector; (2) transferring into Pichia pastoris cells to realize constitutive and secretory expression; (3) fermenting and purifying. The recombinant human proinsulin has such advantages as high stability, high activity and simple production. The method has such advantages as high expression level, high purification yield, and high efficiency.
Description
Technical field
The present invention relates to gene engineering technology field.More specifically, the invention provides a kind of insulin human's original molecule (INS) of novel N-end leader sequence, and a kind of method of utilizing this molecule production proinsulin human, and the structure of related expression carriers and engineering cell, proinsulin human's expression and purifying process.
Background technology
Regular Insulin is the protein hormone of a kind of energy of pancreas excretory lowering blood glucose.During the secretion of Regular Insulin under the normal physiological state be mutually: append secretion and basal secretion after the meal.
Regular Insulin is secreted by B cell.B cell synthesizes a macromolecular preproinsulin earlier, is processed into the proinsulin of 86 peptides then, becomes Regular Insulin through hydrolysis again.
Mature insulin is made of two polypeptide chains of A, B, contain 51 amino acid altogether, about 6000 dalton of molecular weight, less A chain is made up of 21 amino acid, bigger B chain is made up of 30 amino acid, two interchains are connected by two disulfide linkage, destroy the biological action forfeiture that disulfide linkage can make Regular Insulin.The about 5min of serum half-life of Regular Insulin in the human body.
Normal people's Regular Insulin is by liver, kidney degraded, and main degrading enzyme is a liver gsh Regular Insulin desaturase, and this enzyme is inactivation by Regular Insulin being degraded to single A chain and B chain.
Regular Insulin is the specific medicament commonly used of treatment type i diabetes, and the very big market requirement is arranged, and mainly is to extract from pig, Pancreas Bovis seu Bubali in the past, and the recombinant human insulin who produces based on intestinal bacteria (E.coli) has captured most of market share in recent years.
Nineteen twenty-one, Banting and Best successfully extract Regular Insulin from animal pancreatic, have started the history of human insulin's treatment.Be raw material with the pork insulin end of the seventies, adopts the amino-acid substitution technology to obtain the insulin human.Nineteen eighty-two, the insulin human's official listing that adopts gene recombination technology to produce.The nineties, recombinant human insulin's analogue was was successfully researched and developed listing (aminoacid sequence to the insulin human is modified), comprises ultrashort effect and Depot H Insulin's analogue so far.Recombinant human insulin (Recombinant Human Insulin)---be the biotechnology medicine that the applying gene recombinant technology is produced, have structure and the biologic activity identical with human endogenous Regular Insulin.
Proinsulin and analogue gene thereof are a lot of expression systems such as intestinal bacteria, Bacillus subtilus, all obtained expression in yeast and the mammalian cell, the expression amount that has is also than higher, but there is variety of issue, such as intestinal bacteria, expression amount is all right, but expression product often forms inclusion body, though this helps efficiently expressing with stable of product, but these products need increase the complicacy of aftertreatment, and activated to the end efficiency of pcr product be low through becoming renaturation to recover its natural structure and active processing.
The problem that the comprehensive insulin expression production now of the present invention runs into, current research in conjunction with modern gene recombination technology, a kind of insulin human's original molecule of novel N-end leader sequence is provided, and provide a kind of this proinsulin molecule that utilizes, adopt novel Pichia anomala expression system to produce proinsulin human's method, being about to yeast alpha factor leader peptide sequences and new-type human insulin original molecule provided by the invention merges, import a primary yeast constitutive expression carrier, change pichia spp again over to, realize the efficient secretory expression of recombinant insulinum primary, can simplify purifying process greatly, and, obtain fast by optimization for fermentation technology, easy, stable, a kind of proinsulin human's that activity is high production method.
Summary of the invention
Purpose of the present invention just provides a kind of insulin human's original molecule (INS) of novel N-end leader sequence.
Another object of the present invention just provides a kind of method of utilizing the production proinsulin human of this new-type human insulin original molecule high-efficient simple.
Another object of the present invention is exactly expression vector and the engineering cell that is provided for this method.
In a first aspect of the present invention, just provided a kind of insulin human's original molecule of novel N-end leader sequence, it is characterized in that, aminoacid sequence shown in the nucleotide sequence coded SEQ ID of the proinsulin human's molecule NO:2 of described novel N-end leader sequence, and nucleotides sequence is shown 95% above homogeny shown in proinsulin human's molecular core nucleotide sequence coding region of described novel N-end leader sequence and the SEQID NO:1.
In another preference, proinsulin human's molecular core nucleotide sequence of described novel N-end leader sequence contains the nucleotide sequence shown in the SEQ ID NO:1.
In a second aspect of the present invention, a kind of expression vector is provided, it is characterized in that described expression vector contains the described fusion rotein sequence of claim 2.
In another preference, described expression vector is pGAPZA/ α-INS.
In a third aspect of the present invention, a kind of engineering cell is provided, it is characterized in that it is integrated with the described expression vector of claim 4.
In another preference, described engineering cell is a pichia spp.
In a fourth aspect of the present invention, a kind of method of producing recombinant insulinum primary is provided, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 6, thereby secreting, expressing goes out recombinant insulinum primary;
B) separation and purification excretory recombinant insulinum primary.
Description of drawings
Fig. 1 is the structure synoptic diagram of fusion gene α-INS.
Fig. 2 is that recombinant plasmid pGAPZA/ α-INS makes up synoptic diagram.
Embodiment
The inventor is extensive studies by going deep into, a kind of insulin human's original molecule of novel N-end leader sequence is provided, by outside born of the same parents, having made up proinsulin human's molecule fusion protein gene of the novel N-end leader sequence that contains yeast saccharomyces cerevisiae α-factor leader peptide sequences, be built into the Yeast expression carrier that contains the GAP promotor, change pichia spp again over to, make its middle composing type efficient secretory expression in the pichia spp cell.Finished the present invention on this basis.
Natural mature peptide sequence according to the insulin human, consider aftertreatment technology, a kind of insulin human's original molecule of novel N-end leader sequence has been proposed, connect with the Ala-Ala-Asp that can not influence the Regular Insulin natural structure between its B chain and the A chain, and at its N end 6XHisTag label of introducing and a proteolytic enzyme restriction enzyme site, promptly form the F-B-Ala-Ala-Asp-A molecule, wherein F is HHHHHHDDDDK, its complete sequence is seen the aminoacid sequence shown in the SEQ ID NO:2, aminoacid sequence shown in its nucleotide sequence coded SEQ ID NO:2 is shown homology more than 95% with the nucleotides sequence shown in the SEQID NO:1.
The gene order of insulin human's original molecule of full gene synthesizing new N-end leader sequence, with this gene clone in the pUC19 after the sequence verification, use molecular biology method, the fusion sequence of external structure fusion rotein α-INS is cloned into expression vector pGAPZA then then.Transform, be integrated into the P.pastoris host cell chromosome,, screen the strongest clone of resistance, and then filter out the high expression level engineering cell by applying the different concns resistance.Shake flask test shows, grows after 48 hours, more than the expression level 20mg/L.
After obtaining engineering cell, just can be under the condition that is fit to the culturing engineering cell.In the present invention, the recombinant insulinum primary fermentation condition of engineering bacterium expression is not particularly limited.Can adopt the fermentation condition of this area routine.For example, suitable medium includes, but is not limited to following composition:
(i) nitrogenous source contains organic nitrogenous source and inorganic nitrogen-sourced, wherein organic nitrogen source such as peptone, yeast powder, or the mixture of the two, total concn 0.1-3%; Inorganic nitrogen-sourced as ammoniacal liquor or NH
4Cl, (NH
4)
2SO
4Deng ammonium salt, concentration 0-1.5%.
(ii) inorganic salt comprise phosphoric acid salt, Chinese holly hydrochloride, Mg
2+Salt etc.Preferably, phosphate buffer concentration 20-200mM, pH5-8; Mg
2+Salt 0-5mM.
(iii) in substratum, add VITMAIN B1 VITAMIN as a supplement, concentration 1～1000PPM.
(iv) according to M
9Trace element formula in the substratum, trace element can add 0.1-2ml/L training liquid.
(v) carbon source comprises glycerine, glucose, lactose etc.Can be single carbon source, also can be mixed carbon source.Preferably, carbon source is selected from down group: glycerol concentration is 0.1-3%; Lactose concn is 0.1-3%; Glucose concn is 0.1-1.5%.
For the extensive recombinant insulinum primary that obtains, need in fermentor tank, be optimized cultivation.The present invention has studied pilot scale fermentation technology, and the expression level after the optimization reaches 200mg/L.
Behind the fermentation expression recombinant insulinum primary, the rINS that expresses is separated.
Usually, fermented sample earlier obtains fermented liquid supernatant in modes such as centrifugal, filtrations, removes thalline.Fermented liquid supernatant can by saltout, method such as ultrafiltration carries out carrying out chromatography purification again behind the preliminary purification, also can directly carry out chromatography purification.
Be applicable to that chromatographic technique of the present invention comprises metal chelate chromatography, cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography etc.
Through 2-3 step purifying, can obtain the pure product of rINS, the purifying yield is more than 40%, and purity is more than 95%, the about 80mg/L fermented supernatant fluid of pure product yield.
The amount of rINS is exempted from determination of activity and is measured with related kit with putting behind the purifying, is standard substance with the pork insulin.
In an example of the present invention, the α-acquisition of INS antigen-4 fusion protein gene and the structure of expression plasmid are provided.
In another example of the present invention, obtained the bacterial strain of high expression level recombinant insulinum primary by screening, improved the expression amount of recombinant insulinum primary.
In another example of the present invention,, select suitable expression vector express recombinant proinsulin human by the comparison between the different promoters.
In another example of the present invention, the optimization of technology has by fermentation further improved the expression amount of recombinant insulinum primary.
In another example of the present invention,, can obtain pure product 80mg/L through purifying process optimization.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid can obtain the pure product 80mg of recombinant insulinum primary.Be fit to industrialization production.
The invention has the advantages that:
(1) a kind of insulin human's original molecule (INS) of novel N-end leader sequence, and before this sequence, imported yeast saccharomyces cerevisiae α-factor leader peptide sequences, this antigen-4 fusion protein gene is highly suitable for secreting, expressing in the pichia spp, has the characteristics of high expression level, high stable.
(2) expression process is simple; expression vector contains the promotor that does not need methanol induction; can composing type efficiently express the proinsulin human; need not to change substratum during the fermentation or add inductor; simplified operating procedure; have higher security than methyl alcohol as the expression bacterium of inductor, help large-scale production.
(3) by the crucial technological condition for fermentation of control, expression level is further improved.
(4) purifying process is easy, rate of recovery height.Owing to be secretory protein, and have the HisTag label, simplified purification procedures greatly, the purifying rate of recovery is improved greatly, making large-scale low-cost produce recombinant insulinum primary becomes possibility.
(5) adopting pichia spp is engineering cell, has kept recombinant human insulin's activity well.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The acquisition of embodiment 1 fusion gene and the structure of expression plasmid
The gene order of insulin human's original molecule (INS) of full gene synthesizing new N-end leader sequence, with this gene clone in the pUC19 after the sequence verification, use molecular biology method, the fusion sequence (see figure 1) of external structure fusion rotein α-INS then, with the plasmid pUC19/rINS that contains the rINS encoding sequence is template, obtains the INS gene that can merge with correct framework with yeast saccharomyces cerevisiae α-factor leader peptide sequences by pcr amplification.Simultaneously, be template with the plasmid pPIC9K (available from Invitrogen company) that contains yeast saccharomyces cerevisiae α-factor leading peptide encoding sequence, pcr amplification obtains α-factor leader peptide sequences.Above-mentioned two kinds of PCR products behind the purifying are digested with restriction enzyme XhoI respectively, two kinds of enzymes are cut product connect.To connect product is that template is carried out pcr amplification, obtains can be used for the fusion gene α-INS of secreting, expressing.
The construction of recombinant plasmid circuit carries out the α-INS gene of synthetic after enzyme cuts processing with EcoRI as shown in Figure 2, and agarose gel electrophoresis reclaims about 0.5kb fragment; Simultaneously plasmid pGAPZA (available from Invitrogen company) is carried out enzyme with EcoRI and cut, reclaim big fragment.Two fragments are connected with the T4DNA ligase enzyme, connect the product conversion and enter bacillus coli DH 5 alpha (available from Promega company).Prepare plasmid in a small amount, cut by enzyme and identify the positive colony that contains α-INS.PGAPZA can efficiently express its multi-copy integration after inserting foreign gene to the karyomit(e) of host bacterium, itself carry a Zeocin resistant gene simultaneously, can be used for the screening of transformant.The GAP promotor that the constructive expression is arranged before the carrier pGAPZA multiple clone site, do not need inductor can be in host bacterium GS115 efficiently expressing exogenous gene.
The screening of embodiment 2 high expression level recombinant insulinum primary engineering strains
The recombinant plasmid pGAPZA/ α-INS that builds is prepared in a large number, linearizing, electroporation transformed host cell P.pastoris GS115, coating contains the high anti-positive colony of YPDS plate screening of different concns microbiotic Zeocin, and carries out little megger and reach the engineering yeast strain that experiment screening obtains the recombinant insulinum primary high expression level and (contain α-INS).
Embodiment 3 selects suitable expression vector
When construction recombination plasmid pGAPZA/ α-INS, we have made up recombinant plasmid pPIC9K/rINS simultaneously, and by the process similar to embodiment 2, we have obtained the engineering yeast strain (containing rINS) of recombinant insulinum primary high expression level.The expression amount of these two engineering yeast strains under the prerequisite of expressing unanimities such as environment, expression time done a comparison, found to contain the expression amount of proinsulin human in α-INS engineering yeast strain apparently higher than the engineering yeast strain that contains rINS.According to the result, we select GAP as the first-selected promotor of expressing enteropeptidase.
| Bacterial strain | The expression amount of time (mg/L) | ||
| 12 | 24 | 36 | |
| The high expression level engineering yeast strain that pGAPZA/ α-INS transforms | 19 | 42 | 80 |
| The high expression level engineering yeast strain that pPIC9K/rINS transforms | 1 | 7 | 16 |
Embodiment 4 adds the influence of different protein protective agents to expression level
Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; In 1: 10 ratio two-stage inoculation in the 1L of 250ml BMGY Erlenmeyer flask, cultivate about 4 ~ 8hr, last jar of fermentation, pH5.0,20 ℃ of temperature, D0＞35%, treat that dissolved oxygen rising back stream adds 50% glycerine and includes 10%CA, or the nutrient solution of 10%Peptone or 10%Tryptone, take a sample behind the 36hr.Sample detection SDS-PAGE and protein content are to determine the optimum protein protective material of induction period.
Embodiment 5 recombinant insulinum primary purifying
The bacterial cell disruption that will ferment, the centrifuging and taking supernatant places 4 ℃ to prepare chromatographic separation in sample.
Chromatography media: Ni-chelating Sepharose
Chromatography lavation buffer solution: buffer A: 20mM Tris-HCl, 1M NaCl, pH7.4
Buffer B: 20mM Tris-HCl, 0.15M NaCl, pH7.4
Elutriant: contain 500mM Imidazole, the buffer B of 5mM mercaptoethanol
Sample successively with buffer A and buffer B washing, is used elutriant wash-out target protein at last.
Elutriant is handled with enteropeptidase, carries out chromatography then.
Chromatography 2 (sieve chromatography):
Chromatography media: Sephadex G75
Damping fluid: 20mM PB (pH7.0)
Last sample: the sample component that chromatography 1 is collected.
Through above purifying process, can get the pure product 80mg/L of albumen fermented supernatant fluid at last.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.
＜120〉a kind of production method of recombinant insulinum primary
<160>4
<210>1
<211>195
<212>DNA
＜213〉artificial sequence
<221>misc_feature
<222>(1)…(195)
＜223〉insulin human's original molecule (INS) of novel N-end leader sequence
<400>1
catcatcatc atcatcatga cgatgacgat aagtttgtga accaacacct gtgcggctca 60
cacctggtgg aagctctcta cctagtgtgc ggggaacgag gcttcttcta cacacccaag 120
accgctgctg atggcattgt ggaacaatgc tgtaccagca tctgctccct ctaccagctg 180
gagaactact gcaac 195
<210>2
<211>65
<212>PRT
＜213〉homo sapiens
<400>2
His His His His His His Asp Asp Asp Asp Lys Phe Val Asn
1 5 10
Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu
15 20 25
Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala
30 35 40
Ala Asp Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser
45 50 55
Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
60 65
<210>3
<211>255
<212>DNA
＜213〉artificial sequence
<22l>misc_feature
<222>(1)…(255)
＜223〉zymic signal peptide sequence
<400>3
atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60
ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120
tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240
tctctcgaga aaaga 255
<210>4
<211>85
<212>PRT
＜213〉homo sapiens
<400>4
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala
1 5 10
Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp
15 20 25
Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser
30 35 40
Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser
45 50 55
Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile
60 65 70
Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys
75 80
Arg
85
Claims (9)
1. the nucleotide sequence of insulin human's original molecule (INS) of a novel N-end leader sequence, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
2. fusion rotein sequence, it is characterized in that, it contains the described nucleotide sequence of claim 1, and is being inserted with nucleotide sequence shown in SEQ ID NO:3 (aminoacid sequence shown in the SEQ ID NO:3 sequence encoding SEQ ID NO:4) before this nucleotide sequence.
3. an expression vector is characterized in that, it contains the described fusion rotein sequence of claim 2.
4. expression vector as claimed in claim 3 is characterized in that it contains the GAP promotor.
5. an engineering cell is characterized in that, it is integrated with the described expression vector of claim 4.
6. engineering cell as claimed in claim 5 is characterized in that it is a pichia spp.
7. a proinsulin human production method is characterized in that, the method comprising the steps of:
(a) under the expression condition that is fit to, cultivate engineering cell as claimed in claim 6, thereby secreting, expressing goes out proinsulin human's albumen;
(b) separation and purification goes out proinsulin human's albumen of expression.
8. method as claimed in claim 7 is characterized in that, the expression condition shown in the step (a) is: induction time is 12-120 hour, and preferable is 24-100 hour; Inducing pH is 3-9, and that preferable is 5-7.
9. method as claimed in claim 7 is characterized in that, the expression condition shown in the step (b) is:
(1). fermented liquid obtains to contain the supernatant liquor of target protein by simple centrifugal or ultrafiltration;
(2). by easy steps such as simple metal chelate chromatography, hydrophobic chromatographies, can obtain purity at the pure product more than 95%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026290 CN1873006A (en) | 2005-05-30 | 2005-05-30 | Method for producing recombined human proinsulin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026290 CN1873006A (en) | 2005-05-30 | 2005-05-30 | Method for producing recombined human proinsulin |
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| Publication Number | Publication Date |
|---|---|
| CN1873006A true CN1873006A (en) | 2006-12-06 |
Family
ID=37483607
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|---|---|---|---|
| CN 200510026290 Pending CN1873006A (en) | 2005-05-30 | 2005-05-30 | Method for producing recombined human proinsulin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009104199A1 (en) * | 2008-02-19 | 2009-08-27 | Biocon Limited | A method of obtaining purified heterologous insulins expressed in yeast |
| CN105087724A (en) * | 2014-05-04 | 2015-11-25 | 重庆派金生物科技有限公司 | Preparation method for insulin aspart through recombinant expression by using yeast |
| CN105111304A (en) * | 2015-09-30 | 2015-12-02 | 山东阿华生物药业有限公司 | Purification and enzyme digestion transformation method of recombinant human insulin precursor |
| WO2020051812A1 (en) * | 2018-09-12 | 2020-03-19 | 美药星（南京）制药有限公司 | Novel pro-insulin aspart structure and method for preparing insulin aspart |
| CN111094572A (en) * | 2018-02-09 | 2020-05-01 | 江苏恒瑞医药股份有限公司 | Codon-optimized human insulin analogue precursor gene and signal peptide gene |
-
2005
- 2005-05-30 CN CN 200510026290 patent/CN1873006A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009104199A1 (en) * | 2008-02-19 | 2009-08-27 | Biocon Limited | A method of obtaining purified heterologous insulins expressed in yeast |
| CN102015762A (en) * | 2008-02-19 | 2011-04-13 | 百康有限公司 | A method of obtaining purified heterologous insulins expressed in yeast |
| CN102015762B (en) * | 2008-02-19 | 2015-03-18 | 百康有限公司 | A method of obtaining purified heterologous insulins |
| CN105087724A (en) * | 2014-05-04 | 2015-11-25 | 重庆派金生物科技有限公司 | Preparation method for insulin aspart through recombinant expression by using yeast |
| CN105087724B (en) * | 2014-05-04 | 2019-12-03 | 重庆派金生物科技有限公司 | The preparation method of yeast recombinant expression insulin aspart |
| CN105111304A (en) * | 2015-09-30 | 2015-12-02 | 山东阿华生物药业有限公司 | Purification and enzyme digestion transformation method of recombinant human insulin precursor |
| CN105111304B (en) * | 2015-09-30 | 2018-12-14 | 山东阿华生物药业有限公司 | The purifying and digestion conversion method of rh-insulin's precursor |
| CN111094572A (en) * | 2018-02-09 | 2020-05-01 | 江苏恒瑞医药股份有限公司 | Codon-optimized human insulin analogue precursor gene and signal peptide gene |
| CN111094572B (en) * | 2018-02-09 | 2023-04-04 | 江苏恒瑞医药股份有限公司 | Codon-optimized human insulin analogue precursor gene and signal peptide gene |
| WO2020051812A1 (en) * | 2018-09-12 | 2020-03-19 | 美药星（南京）制药有限公司 | Novel pro-insulin aspart structure and method for preparing insulin aspart |
| CN112584853A (en) * | 2018-09-12 | 2021-03-30 | 美药星（南京）制药有限公司 | Structure of novel insulin aspart and method for preparing insulin aspart |
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