CN102628070A - Method for converting expression vector of insulin precursor - Google Patents
Method for converting expression vector of insulin precursor Download PDFInfo
- Publication number
- CN102628070A CN102628070A CN2012101075773A CN201210107577A CN102628070A CN 102628070 A CN102628070 A CN 102628070A CN 2012101075773 A CN2012101075773 A CN 2012101075773A CN 201210107577 A CN201210107577 A CN 201210107577A CN 102628070 A CN102628070 A CN 102628070A
- Authority
- CN
- China
- Prior art keywords
- insulin
- expression vector
- lantus
- affinity resin
- fusion rotein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for converting an expression vector of an insulin precursor. The method comprises the following steps of: inoculating a colon bacillus monoclonal colony of an expression vector plasmid which contains a gene engineering insulin glargine precursor, and centrifugally collecting thallus; dissolving the thallus into a buffer solution, cracking bacteria, collecting an insulin glargine fusion protein inclusion body, and dissolving into a buffer solution containing urea; adding a cobalt ion affine resin into the solution, stirring and mixing uniformly, combining with a long-acting insulin fusion protein, and centrifugally collecting a metal affine resin; and collecting the cobalt ion affine resin, flushing the metal affine resin with a buffer solution of urea repeatedly, eluting the combined long-acting insulin fusion protein with a buffer solution, and sampling respectively for synthesizing a protein electrophoresis detection insulin fusion protein. The method has the advantages of increase in production efficiency and reduction in production cost.
Description
Technical field
The present invention relates to a kind of method of expression vector conversion of insulin precurosor, belong to genetically engineered and technical field of molecular biology.The present invention provides a kind of and is not using chemical inducer and do not adding under any antibiotic condition, obtains efficient, the interior conversion expression of body reliably, and can carry out the method that cultured continuously prepares the protamine zine insulin fusion rotein.
Background technology
Mellitus are a kind of common incretion metabolism diseases.In recent years, mellitus just have swept the globe with unprecedented gesture.In China, along with the change of living standards of the people raising and mode of life, diabetes prevalence raises just fast, and presents the trend of the rejuvenation of falling ill.According to statistics, diabetic subject's number that China has made a definite diagnosis becomes the maximum country of diabetic subject in the world up to 9,240 ten thousand people.The serious harm people ' s health.Acute and chronic complication, the especially chronic complicating diseases of mellitus are disabled, lethality rate is high, have a strong impact on patient's physical and mental health, and bring white elephant for individual, family and society.
In treating diabetes, Regular Insulin is up to standard to controlling blood sugar, complication prevention has irreplaceable effect, also is the most effective one of treating diabetes means at present.In recent years, along with the development of Regular Insulin technology, developed insulin analog.Wherein a kind of Recent Development of Long-acting Insulin Analogs-Lantus is by increasing doctor and patient's acceptance and use.
The reorganization Lantus is the BHI's analogue that utilizes recombinant DNA technology to produce.It is to have increased by two l-arginine at insulin human B chain C-terminal; Simultaneously also replace to glycocoll to the l-asparagine of A chain C-terminal A21 position; After the subcutaneous injection; Because of acidic solution is neutralized the sustainable slow release reorganization of the small deposition that forms Lantus, continue to be absorbed into blood also slow, thereby produce the foreseeable Plasma Concentration that reaches 24 hours steady no peak values.Therefore, every day, regularly subcutaneous injection once can be satisfied the needs of human body to basal insulin.In a word, continuing to improve under the situation of glycemic control, the reorganization Lantus can reduce the hypoglycemia incidence, improves patients ' life quality, does not increase medical expense simultaneously.
On Recombulin; Be two kinds of expression systems at present; One is the inclusion bodies of colibacillus expression system, is mainly gift and comes company and Sanofi-Aventis company to use, and representational insulin product is recombinant human insulin, Insulin lispro and Lantus; This technology is comparatively ripe, and exploitation early.The most outstanding advantage of escherichia expression system is that technology is simple, output is high, the cycle is short, production cost is low.Yet numerous protein needs through the processing of the modification after the translation after translation.Inclusion body is become renaturation and pancreatin and protaminase carried out enzyme cut, carry out consummate through ion exchange chromatography and reversed phase chromatography then.Another kind is a yeast expression system; Being mainly Denmark Novo Nordisk Co.,Ltd adopts; Use has the yeast saccharomyces cerevisiae of posttranslational modification as expression system; Saved the loaded down with trivial details and high flow rate operation steps of inclusion body change renaturation, representational product is recombinant human insulin, insulin aspart and insulin detemir.
We have adopted the inclusion bodies of colibacillus expression system in the research work to recombination Depot H Insulin analogue.Intestinal bacteria are to study the most sophisticated gene engineering expression system, and current business-like gene engineering product is through escherichia coli expression mostly.The production of recombination Depot H Insulin analogue preparation at first needs the expression vector of artificial constructed synthetic Lantus, for the batch process of insulin analog provides the basis.And the crucial purpose of the present invention to be exactly expression vector with the genetically engineered insulin precurosor be transformed into the high expression level intestinal bacteria.
We can find a kind ofly not need with any microbiotic and can carry out the expression method of cultured continuously in R&D process.According to natural human insulin's aminoacid sequence, synthesize and optimized gene DNA fragment, through pcr amplification, respectively product is connected after double digestion is handled with plasmid, form the vector plasmid parent of insulin expression.Pass through genetically engineered again; The synthetic gene of human insulin gene A, B chain is combined to respectively on the colibacillary different plasmid; Move to then in the thalline, duplicate and express, thereby make the engineering strain that has A, B chain gene produce insulin human A, B chain respectively; Use the artificial method again, make these two chains connect into activated insulin human through disulfide linkage external.
Genetically engineered insulin human's primary structure:
Normal (centre) insulin human's of genetically engineered structure is as shown in Figure 1, and the structure of genetically engineered protamine zine insulin is as shown in Figure 2.
Genetically engineered protamine zine insulin precursor-gene sequence:
TTC?GTC?AAT?CAG?CAC?CTT?TGT?GGT?TCT?CAC?CTC?GTT?GAA?GCT?TTG?TAC?CTT?GTT?TGC?GGT?GAA?CGT?GGT?TTC?TTC?TAC?ACT?CCT?AAG?ACT?CGT?CGT?GAA?GCT?GAA?GAC?CTC?CAG?GTT?GGT?CAG?GTT?GAA?CTC?GGT?GGT?GGT?CCT?GGT?GCT?GGT?TCT?CTT?CAA?CCT?CTT?GCT?CTT?GAA?GGT?TCT?CTT?CAG?AAG?CGT?GGC?ATC?GTT?GAA?CAG?TGT?TGC?ACA?TCT?ATC?TGC?TCT CTT?TAC?CAG?CTT GAG?AAC?TAC?TGT?GGT?TAA?TAA
Summary of the invention
The expression vector method for transformation that the purpose of this invention is to provide a kind of insulin precurosor behind the expression vector of the good Regular Insulin of Primary Construction, further is transformed into colibacillary effective means with the expression vector that builds.
For achieving the above object, technical scheme of the present invention provides a kind of expression vector method for transformation of insulin precurosor, and concrete steps are:
A: the intestinal bacteria mono-clonal bacterium colony that will contain the expression vector plasmid of genetically engineered Lantus precursor inserts in the tryptic soy broth; In shaking table, cultivate, the temperature of cultivation is 25-40oC, rotating speed 200-300RPM; Cultivate after 15-30 hour, collect thalline through centrifugation method;
B: the thalline of the centrifugal collection of the first step is dissolved into 10-20 mM Tris damping fluid; Ultrasonic bacterium cracking; Be under the condition of 6000g centrifugal 30-45 minute at 4 ° of C, cf-with lysate then, collect Lantus fusion rotein inclusion body afterwards, the Lantus fusion rotein solubilization of inclusion bodies that it is collected is to containing in the plain 10-20 mM Tris damping fluid of 6-8 M urea; In solution, add metal affinity resin then; Stirring and evenly mixing makes it combine the Lantus fusion rotein, collects metal affinity resin through centrifugation method at last;
C: with the metal affinity resin of the second centrifugal collection of step; With the plain 10-20mM Tris damping fluid flushing metal affinity resin several of 6-8 M urea; To remove impurity; With containing the plain 10-20 mM Tris damping fluid of 100-200 mM imidazoles and 6-8 M urea with bonded Lantus fusion rotein wash-out, the synthetic of protein electrophoresis detection Regular Insulin fusion rotein carried out in sampling respectively at last.
Further improvement of the present invention is: the pH value of the damping fluid that uses in this method is 8-10,
Further improvement of the present invention is: the metal affinity resin among the step B is cobalt ion or nickel ion affinity resin.
The present invention is initial target with the expression vector of insulin-containing precursor at first, is transformed into intestinal bacteria through expression vector; A large amount of high-efficient clonings in intestinal bacteria then, thus can access a large amount of Recombulins.The comprehensive above material that just can screen the insulin-containing precursor of reliable high expression level.
The present invention has the following advantages: any microbiotic can not need be used in (1), production process: the bacterium that will contain the expression vector plasmid is divided into two parts and is inoculated into the cultivation of tryptic soy broth shaking table; Contain 50 μ g/ml kantlex a the adding, and another part does not add any microbiotic.The result finds almost do not have much difference at the expression amount that does not use recombination fusion protein under the antibiotic condition with using antibiotic cultivation.This shows that this expression system is highly stable in the intestinal bacteria body, helps the simplification of production technique;
(2), expression vector is stable, can carry out cultured continuously: will carry Lantus gene plasmid intestinal bacteria antibiotic-free and cultivate, cultured continuously 2 days, plasmid does not almost have to be lost, cultured continuously 4 days, the amount of bacteria that contains plasmid still accounts for more than 60%.The Recombinant Protein Expression amount is directly proportional with the amount of bacteria that contains plasmid;
(3), need not use chemical inducer; The microbionation that will contain the expression vector plasmid is divided into two parts of cultivations to the tryptic soy broth shaking table, a 1mMIPTG inductor that adds, and another part does not add inductor.Sampling is carried out protein electrophoresis and is detected the synthetic of Regular Insulin fusion rotein, and the result finds: the cultivation that does not add inductor can be synthesized a large amount of Regular Insulin fusion roteins as usual.Although add per-cent that the cultivation Regular Insulin fusion rotein resultant quantity of inductor accounts for total bacterial protein slightly more than the cultivation of not using inductor; But because the use of inductor has suppressed the growth of bacterium greatly, its unit volume nutrient solution Regular Insulin fusion rotein resultant quantity is well below the cultivation of not using inductor.These characteristics not only might be simplified production technique, and can not need expensive chemical inducer, thereby can enhance productivity, and reduce production costs.
Embodiment
In order to deepen to understanding of the present invention, will combine embodiment that the present invention is made further detailed description below, this embodiment only is used to explain the present invention, does not constitute the qualification to protection domain of the present invention.
Embodiment 1:
A kind of expression vector method for transformation of insulin precurosor, concrete steps are:
A: the intestinal bacteria mono-clonal bacterium colony that will contain the expression vector plasmid of genetically engineered Lantus precursor inserts in the tryptic soy broth; In shaking table, cultivate, the temperature of cultivation is 25-40oC, rotating speed 200-300RPM; Cultivate after 15-30 hour, collect thalline through centrifugation method;
B: the thalline of the centrifugal collection of the first step is dissolved into 10-20 mM Tris damping fluid; Ultrasonic bacterium cracking is under the condition of 6000g centrifugal 30-45 minute at 4 ° of C, cf-with lysate then, collects Lantus fusion rotein inclusion body afterwards; The Lantus fusion rotein solubilization of inclusion bodies that it is collected is to containing in the plain 10-20 mM Tris damping fluid of 6-8 M urea; In solution, add metal affinity resin then, metal affinity resin is cobalt ion or nickel ion affinity resin, stirring and evenly mixing; Make it combine the Lantus fusion rotein, collect metal affinity resin through centrifugation method at last;
C: with the metal affinity resin of the second centrifugal collection of step; With the plain 10-20mM Tris damping fluid flushing metal affinity resin several of 6-8 M urea; To remove impurity; With containing the plain 10-20 mM Tris damping fluid of 100-200 mM imidazoles and 6-8 M urea with bonded Lantus fusion rotein wash-out, the synthetic of protein electrophoresis detection Regular Insulin fusion rotein carried out in sampling respectively at last.
Wherein, The pH value of the damping fluid that uses among the present invention is 8-10; The invention provides a kind of expression vector method for transformation of insulin precurosor; The intestinal bacteria mono-clonal bacterium colony that will contain the expression vector plasmid of genetically engineered Lantus precursor inserts in the tryptic soy broth cultivates abduction delivering, centrifugal then collection thalline; The thalline dissolving of collecting, the cracking bacterium, centrifugal collection Lantus fusion rotein inclusion body with the inclusion body dissolving, adds metal affinity resin, stirring and evenly mixing, centrifugal collection metal affinity resin then; The metal affinity resin Tris damping fluid flushing of collecting is removed impurity for several times, with containing the plain Tris damping fluid of imidazoles and urea with bonded Lantus fusion rotein wash-out; Sample thief carries out protein electrophoresis and detects, and verifies that this albumen is desirable proteins.The present invention adopts the vitro culture mode, do not contain microbiotic, without chemical inducer, good stability, expression amount is high, application prospect is wide.
Claims (3)
1. the expression vector method for transformation of an insulin precurosor, it is characterized in that: concrete steps are:
A: the intestinal bacteria mono-clonal bacterium colony that will contain the expression vector plasmid of genetically engineered Lantus precursor inserts in the tryptic soy broth; In shaking table, cultivate, the temperature of cultivation is 25-40oC, rotating speed 200-300RPM; Cultivate after 15-30 hour, collect thalline through centrifugation method;
B: the thalline of the centrifugal collection of the first step is dissolved into 10-20 mM Tris damping fluid; Ultrasonic bacterium cracking; Be under the condition of 6000g centrifugal 30-45 minute at 4 ° of C, cf-with lysate then, collect Lantus fusion rotein inclusion body afterwards, the Lantus fusion rotein solubilization of inclusion bodies that it is collected is to containing in the plain 10-20 mM Tris damping fluid of 6-8 M urea; In solution, add metal affinity resin then; Stirring and evenly mixing makes it combine the Lantus fusion rotein, collects metal affinity resin through centrifugation method at last;
C: with the metal affinity resin of the second centrifugal collection of step; With the plain 10-20mM Tris damping fluid flushing metal affinity resin several of 6-8 M urea; To remove impurity; With containing the plain 10-20 mM Tris damping fluid of 100-200 mM imidazoles and 6-8 M urea with bonded Lantus fusion rotein wash-out, the synthetic of protein electrophoresis detection Regular Insulin fusion rotein carried out in sampling respectively at last.
2. according to the expression vector method for transformation of the said a kind of insulin precurosor of claim 1, it is characterized in that: the pH value of the damping fluid that uses in this method is 8-10.
3. according to the expression vector method for transformation of the said a kind of insulin precurosor of claim 1, it is characterized in that: the metal affinity resin among the said step B is cobalt ion or nickel ion affinity resin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101075773A CN102628070A (en) | 2012-04-13 | 2012-04-13 | Method for converting expression vector of insulin precursor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012101075773A CN102628070A (en) | 2012-04-13 | 2012-04-13 | Method for converting expression vector of insulin precursor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102628070A true CN102628070A (en) | 2012-08-08 |
Family
ID=46586462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012101075773A Pending CN102628070A (en) | 2012-04-13 | 2012-04-13 | Method for converting expression vector of insulin precursor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102628070A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589767A (en) * | 2012-08-17 | 2014-02-19 | 宜昌长江药业有限公司 | Application of arginine in improving expression quantity of fermentation-cultured polypeptide containing arginine-arginine in amino acid sequence and method |
| CN113773392A (en) * | 2020-06-09 | 2021-12-10 | 宁波鲲鹏生物科技有限公司 | Preparation method of insulin glargine |
-
2012
- 2012-04-13 CN CN2012101075773A patent/CN102628070A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103589767A (en) * | 2012-08-17 | 2014-02-19 | 宜昌长江药业有限公司 | Application of arginine in improving expression quantity of fermentation-cultured polypeptide containing arginine-arginine in amino acid sequence and method |
| CN103589767B (en) * | 2012-08-17 | 2016-03-02 | 宜昌东阳光长江药业股份有限公司 | Arginine contains application in the expression amount of the polypeptide of Arg-Arg and method improving fermentation culture aminoacid sequence |
| CN113773392A (en) * | 2020-06-09 | 2021-12-10 | 宁波鲲鹏生物科技有限公司 | Preparation method of insulin glargine |
| CN113773392B (en) * | 2020-06-09 | 2023-04-07 | 宁波鲲鹏生物科技有限公司 | Preparation method of insulin glargine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102618552B (en) | Productive technology of recombined exenatide | |
| CN104402975B (en) | Anti-aging small peptide and preparation method thereof | |
| CN1850964A (en) | Highactive wild Chinese caterpillar fungus strain, and its fruiting body and artificial culture method | |
| CN101104637A (en) | Cecropin and its generation bacterial strain and use | |
| CN104844684A (en) | Protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier | |
| CN102628070A (en) | Method for converting expression vector of insulin precursor | |
| CN101525387A (en) | Recombined long-acting glucagons peptide analogue and preparation method thereof | |
| CN107937408A (en) | Epinephelus coioides insulin genes, encoding proteins and its application | |
| CN115011622B (en) | Screening method and application of D-psicose 3-epimerase mutant | |
| CN102898512B (en) | Recombinant plectasin as well as preparation method and application of recombinant plectasin | |
| CN101979572B (en) | Preparation for South China Sea conus striatus toxin S4.3 and application | |
| CN108948163A (en) | Queensland nut plant alexin and its application | |
| CN112625925B (en) | High-yield strain of dalbavancin precursor A40926B0 and application thereof | |
| CN104558148A (en) | Ciliary neurotrophic factor mutant, and modified mutant and application thereof | |
| CN104418945A (en) | Preparation method of peptide and application of peptide in preparation of medicine and feed additive | |
| CN101948837A (en) | Method for producing human insulin growth factor-1 in recombinant Escherichia coli | |
| CN103045631B (en) | Preparation method for oral hypoglycemic recombinant human proinsulin | |
| CN102260346B (en) | Exendin-4 analog | |
| CN108265068B (en) | Recombinant arginine deiminase and industrial preparation method and application thereof | |
| CN102070713B (en) | Process for purifying Exendin-4 in Esherichia coli through one-step chromatography | |
| CN103833828B (en) | Extraction method of insulin glargine precursor protein | |
| CN103409444B (en) | CDNA (Complementary Deoxyribose Nucleic Acid) nucleotide of monascus ruber GAD (Glutamic Acid Decarboxylase) gene and synthetic method thereof and corresponding protein | |
| CN100523172C (en) | Gene engineering bacteria of high efficiency expression of human alpha 1-thymulin and its construction method and use | |
| CN102465129A (en) | Solubility expression and preparation of human fibroblast growth factor 21 in procaryotic cell | |
| TWI521060B (en) | A method for culturing the larvae of Cordyceps sinensis, a composition comprising Cordyceps sinensis and its use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120808 |