CN111662836A - Genetically engineered bacterium for expressing insulin precursor and preparation method and application thereof - Google Patents
Genetically engineered bacterium for expressing insulin precursor and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a genetically engineered bacterium for expressing an insulin precursor, which is an engineered bacterium integrating an expression vector of an insulin precursor gene in pichia pastoris, wherein the nucleotide sequence of the insulin precursor gene is shown as SEQ ID No.1 in a sequence table. The invention also provides a preparation method of the insulin precursor. The strain yield of the genetically engineered bacteria fermented in the shake flask level is higher than that reported in the prior art. The yield of the insulin precursor obtained by fermentation in a horizontal fermentation tank by utilizing the genetically engineered bacteria and the preparation method of the insulin precursor is remarkably improved. In addition, the culture medium used in the preparation method of the insulin precursor has low cost, does not have the risk of animal-derived virus pollution, and obviously reduces the cost of the preparation method.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a genetic engineering bacterium for expressing an insulin precursor, a preparation method and application thereof, and a preparation method of the insulin precursor.
Background
Diabetes is a chronic metabolic disease. Due to aging and rapid growth of the population, the number of diabetic patients may increase by 50.7% globally by 2030. Recombinant human insulin and its analogs are currently the most effective and major treatment for diabetes. The mainstream insulin-like medicaments in the market comprise insulin aspart, insulin glargine, insulin detemir, insulin deglutamide and the like, wherein the last three are long-acting insulin, the action in a human body can last for 24 hours, and no concentration peak exists. The existing technology of insulin analogs mostly adopts an escherichia coli system and a yeast system to prepare a precursor, and then the precursor is converted into human insulin or side chain modification is carried out to obtain the long-acting insulin. Insulin analogs herein refer to analogs obtained by amino acid position changes in human insulin, such as insulin glargine, insulin aspart, DesB30, and the like.
The pichia system is commonly used in the prior art to express insulin precursors such as DesB30 precursor. The pichia pastoris has the advantages of cheap culture medium, fast growth, easy purification and the like. Under the regulation and control of the alcohol oxidase promoter, the exogenous protein can be efficiently expressed without excessive glycosylation. DesB30 lacks threonine at position 30 in the B chain compared to human insulin. The product DesB30 precursor of the expression supernatant is subjected to enzyme digestion to obtain DesB 30. The present literature reports on the Expression of DesB30 Precursor (Xie T, LiuQ, Xie F, et al. secretion Expression of Insulin Precursor in Pichia pastoris and Simple Procedure for Producing Recombinant Human Insulin [ J ]. Preparative biochemistry,2008,38(3): 308. 317; Gurramkonda C, Polez S, Skoo N, et al. application of Simple fed-batch detection to high-level secretion of Insulin resistor using Pichia pastoris with purification and conversion to Human Insulin [ J ]. Microcell industries, 9(1):31-42. 21. 3.84. high density fermentation/3. g. In the literature, the effect of different promoters on high-density expression of human insulin precursor by recombinant pichia pastoris [ D ]. Chongqing: when Pichia pastoris was cultured with BMGY at Chongqing university of Rich, 2012, the IP yield could be 90mg/L at the shake flask level. Therefore, a strain expressing insulin precursor with high yield is urgently needed.
The prior art references relating to reducing the salt concentration of BSM media include, for example, ZHao H L, Xue C, Wang Y, et al, incorporated the cell viability and heterologous protein expression of Pichia pastoris microorganism by culture regulation [ J ]. Appl Microbiol Biotechnology, 2008,81(2): 235-241. The expression quantity of the fusion protein reaches 0.68g/L after the HSA-IFN-alpha 2b fusion protein is used as a target protein and optimized through various conditions (including the salt concentration of a culture medium, the culture temperature, the prolonged production period and the like), and the expression quantity reaches 1.26g/L after peptone is added into the culture medium. In addition, the initial concentration of the target protein is lower, and the yield of the target protein can be increased correspondingly easily after optimizing the conditions.
Disclosure of Invention
The invention aims to overcome the defects that the yield of the insulin precursor of a strain expressing the insulin precursor at the level of a shake flask or a fermentation tank is low, a culture medium used in the production process is expensive, animal-derived viruses are easy to introduce and the like in the prior art, and provides a genetically engineered bacterium for expressing the insulin precursor, a preparation method and application thereof, and a preparation method of the insulin precursor. The strain yield of the genetically engineered bacteria fermented in the shake flask level is higher than that reported in the prior art. The yield of the insulin precursor obtained by fermentation in a horizontal fermentation tank by utilizing the genetically engineered bacteria and the preparation method of the insulin precursor is remarkably improved. In addition, the culture medium used in the preparation method of the insulin precursor has low cost, does not have the risk of animal-derived virus pollution, obviously reduces the cost of the preparation method, and is beneficial to industrial production.
The invention effectively improves the yield of the insulin precursor obtained by the genetic engineering bacteria which integrates the gene sequence through the optimization of the gene sequence of the insulin precursor in the horizontal fermentation of a shake flask (the yield of the insulin precursor obtained in a preferred embodiment of the invention is 136mg/L, but the yield is 90mg/L in the existing report). In addition, because the expression level of the exogenous protein is related to a plurality of factors, including the specificity of a strain, the nature of the exogenous protein, the components of a culture medium, a culture environment, a fermentation process and the like, a plurality of experimental schemes are provided for improving the expression level of the exogenous protein, the inventor unexpectedly discovers that the yield of the originally expressed insulin precursor is high when the BSM culture medium with reduced salt concentration is used for fermenting and culturing the Pichia pastoris engineering bacteria (Pichia pastoris), and finally discovers that the yield of the obtained insulin precursor is further remarkably improved by reducing the osmotic pressure of the culture medium, and the direct relation between the osmotic pressure of the culture medium and the improvement of the yield of the insulin precursor is firstly discovered. The art has used BSM with reduced salt concentrations to culture fermenting yeast strains to increase their production of expressed foreign proteins, all starting from lower initial concentrations. However, the inventor starts from a strain expressing a target protein with a high initial concentration (3.31g/L) in one embodiment of the invention through optimization of a technical scheme, and the yield of the target protein expressed by the strain is remarkably increased to 4.51g/L after final optimization, which is not easy in the field.
In order to solve the technical problem, the invention provides a genetically engineered bacterium for expressing an insulin precursor, which is an engineered bacterium integrating an expression vector of an insulin precursor gene in Pichia pastoris (Pichia pastoris), wherein the nucleotide sequence of the insulin precursor gene is shown as SEQ ID No.1 in a sequence table.
Preferably, the pichia pastoris is pichia pastoris GS 115.
Preferably, the backbone of the expression vector is plasmid pPIC3.5, pPIC9K or pPICZ alpha.
Preferably, the expression vector contains AOX1 promoter.
Preferably, the expression vector has an anti-geneticin gene, and the genetically engineered bacterium has the performance of resisting geneticin with the concentration of less than 5.0mg/mL, preferably has the performance of resisting geneticin with the concentration of 0.25-5.0 mg/mL.
In order to solve the technical problems, the invention provides a gene for coding an insulin precursor, and the nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table.
In order to solve the above technical problems, the present invention provides an expression vector having the above gene encoding insulin precursor integrated therein.
Preferably, the backbone of the expression vector is plasmid pPIC3.5, pPIC9K or pPICZ alpha.
Preferably, the expression vector contains AOX1 promoter.
Preferably, the expression vector has an anti-geneticin gene, preferably an anti-geneticin gene with a concentration of less than 5.0mg/mL, more preferably an anti-geneticin gene with a concentration of 0.25-5.0 mg/mL.
In order to solve the technical problems, the invention provides a preparation method of the genetically engineered bacterium, which comprises the following steps:
(1) constructing the expression vector;
(2) transforming the expression vector obtained in the step (1) into Pichia pastoris;
(3) and (3) coating the pichia pastoris obtained in the step (2) on a plate containing geneticin for culture, and then selecting transformants on the plate.
Preferably, the conversion in step (2) is electric conversion, and the electric potential of the electric conversion is preferably 0.75-1.5 Kv/cm, and more preferably 1.1 Kv/cm.
Preferably, the concentration of geneticin in step (3) is less than 5.0mg/mL, preferably 0.25-5.0 mg/mL, for example 5.0 mg/mL.
Preferably, the plate in step (3) is a YPD plate.
Preferably, the culture condition in the step (3) is 22-30 ℃; preferably 28 deg.c.
In order to solve the technical problems, the invention provides a preparation method of an insulin precursor, which comprises the steps of inoculating the genetic engineering bacteria into a pichia pastoris culture medium for fermentation, and obtaining the insulin precursor from fermentation liquor.
Preferably, the fermentation is conventional in the art and may be in a fermentor, for example typically in a 3L, 5L or 10L fermentor.
Preferably, the culture medium of the pichia pastoris is a BSM culture medium, the concentration of the other components except the glycerol and the pichia pastoris microelement 1 is reduced to 1/2 at most, and preferably to 1/2.
More preferably, the reduced-to-1/2 BSM medium (i.e., 1/2BSM medium in the prior art) comprises 4% glycerol, 1.335% phosphoric acid, 0.0465% calcium sulfate dihydrate, 0.91% potassium sulfate, 0.745% magnesium sulfate heptahydrate, 0.206% potassium hydroxide, and 0.4% pichia pastoris trace element 1(PTM 1); the phosphoric acid and the pichia pastoris microelement 1 are in percentage by volume of each component in the culture medium; the percentage of the other components is the mass volume percentage of each component in the culture medium.
More preferably, the pichia pastoris trace element 1 preferably comprises 0.6% copper sulfate pentahydrate, 0.008% sodium iodide, 0.3% manganese sulfate monohydrate, 0.02% dihydrate and sodium molybdate, 0.002% boric acid, 0.05% cobalt chloride, 2% zinc chloride, 6.5% ferrous sulfate heptahydrate, 0.02% biotin and 0.5% sulfuric acid; the percentage of the sulfuric acid is the volume percentage of the sulfuric acid in the pichia pastoris microelement 1; the percentage of the other components is the mass volume percentage of each component in the pichia pastoris microelement 1.
In the present invention, the mass volume percentage may be g/ml as is conventional in the art, unless otherwise specified.
Preferably, in the fermentation culture process, 0.5-2.0% by volume of methanol is added every 12-24 hours; methanol is preferably added in a volume fraction of 1% every 12 hours.
In order to solve the technical problems, the invention provides the application of the genetically engineered bacterium, the gene or the expression vector in preparing the insulin precursor.
In order to solve the technical problems, the invention provides the application of the BSM culture medium in preparing the insulin precursor by biological fermentation, wherein the concentration of the components except the glycerol and the pichia pastoris microelement 1 is reduced to 1/2.
Preferably, the BSM medium reduced to 1/2 is the BSM medium reduced to 1/2, and/or the biological fermentation is fermentation by using the genetically engineered bacterium expressing insulin precursors.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the strain yield of the genetically engineered bacteria fermented in the shake flask level is higher than that reported in the prior art. In a preferred embodiment of the present invention, the yield of insulin precursor is 136mg/L, which is reported to be 90 mg/L. The yield of the insulin precursor obtained by fermentation in a horizontal fermentation tank by utilizing the genetically engineered bacterium provided by the invention in combination with the preparation method of the insulin precursor provided by the invention is remarkably improved. In a preferred embodiment of the present invention, 1/2BSM medium, optimized for yeast Basal Salts Medium (BSM), achieved an IP yield of 4.51g/L after 156 hours of induction in a 5L fermentor. In addition, the culture medium used in the preparation method of the insulin precursor has low cost, does not have the risk of animal-derived virus pollution, obviously reduces the cost of the preparation method, and is beneficial to industrial production.
Drawings
FIG. 1 is a schematic diagram of the construction of pPIC 9K-IP.
FIG. 2 is a schematic diagram showing control expression detection of randomly selected 10 transformants on MD plates and empty plasmids; wherein, M is a protein Marker: 31kDa, 20.1kDa, 14.4kDa, 6.5kDa, 3.3 kDa; 1 is pPIC9K empty vector transformation bacteria expression supernatant; 2-11 are the expression supernatants of 10 transformants in MD plates, respectively. The loading amount was 10. mu.L.
FIG. 3 is a diagram showing the measurement of the expression level of transformants in YPD plates containing G418 at different concentrations; wherein, M is a protein Marker: 31kDa, 20.1kDa, 14.4kDa, 6.5kDa, 3.3 kDa; 1-5 at 5mg/mL G418, 6-8 at 6mg/mLG418, and 9-10 at 7mg/mL G418, respectively, correspond to transformants in the plate. The loading amount was 10. mu.L.
FIG. 4 shows the effect of different methanol additions on biomass and IP expression during the induction period of the shake flask level.
FIG. 5 is a diagram showing the measurement of the expression level in a 5L fermenter, in which M is a protein Marker; 1-3 are fermentation supernatants after induction of the BSM culture medium for 108, 120 and 132 hours respectively; 4-6 are fermentation supernatants after induction of 1/2BSM medium for 132, 144, and 156 hours, respectively. The loading was 5. mu.L.
FIG. 6 is a schematic diagram showing the relationship between the wet weight of the cells (WCW), the concentration of the target protein, and the osmotic pressure of the medium at the level of the 5L fermentor and the fermentation time.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1: construction and screening of expression vector pPIC9K-IP (IP is insulin precusor, and plasmid map is shown in figure 1) and Pichia pastoris engineering bacteria
The invention adopts pPIC9K vector, cloning sites are EcoR I and Not I, and recombinant vector is pPIC 9K-IP. The expression vector initiates transcription from the AOX1 promoter.
A target gene fragment (the specific sequence of the gene sequence is shown in SEQ ID NO.1) was synthesized by Shanghai Ruidi Biotech Co., Ltd and cloned on pPIC9K vector (purchased from Invitrogen) at EcoR I and NotI sites to obtain plasmid pPIC 9K-IP.
2. The plasmid pPIC9K-IP obtained above was extracted and the plasmid linearized digestion was carried out using the following digestion system:
enzyme cutting is carried out for 4 hours at 37 ℃, and the enzyme cutting product is placed at 70 ℃ for 10 minutes to inactivate the restriction enzyme; and (3) recovering the enzyme digestion product by using an ethanol precipitation recovery method to obtain a linearized plasmid recovered product, wherein a nano drop detection result shows that the concentration of the recovered linearized plasmid is about 1000 ng/mu L.
3. Preparation and electrotransformation of pichia pastoris competent cells
(1) The Pichia GS 115-expressing strain (purchased from Invitrogen) was inoculated into 5mL of YPD liquid medium (composition: 10g/L yeast extract, 20g/L tryptone, 20g/L glucose) and cultured at 28 ℃ and 230rpm for about 18 hours to OD600 of 1.2-1.5;
(2) subpackaging 5mL of the GS115 culture solution obtained in the step (1) into 5 sterile EP tubes of 1.5mL, wherein each tube contains 1mL of culture solution, and centrifuging at the rotating speed of 1500g for 30 seconds;
(3) discarding the supernatant, resuspending each tube of the bacteria in 800. mu.L of ice-cold sterile water, and centrifuging at 1500g for 30 s;
(4) repeating the step (3) once;
(5) the supernatant was discarded, and each tube of the cells was resuspended in 400. mu.L of ice-cold sterile sorbitol (1M, sigma-adrich), and centrifuged at 1500g for 30 seconds;
(6) repeating the step 5 once;
(7) the supernatant was discarded, each tube of the cells was resuspended in 16. mu.L of ice-cold sterile sorbitol (1M), and the cells from 5 EP tubes were mixed, i.e., 80. mu.L of competent GS115 cells.
(8) Taking 8 mu L of the recovered linearized plasmid, adding the recovered linearized plasmid into the competent GS115 cell prepared in the step (7), and carefully blowing the recovered product by using a pipette gun to fully and uniformly mix the linearized plasmid and the competent cell; after transformation with an electric shock, 1mL of ice-cold sorbitol (1M) was added thereto at a voltage of 1.1kV, and the mixture was incubated at 28 ℃ for 2 hours at rest, and MD plates (composed of g/L glucose, 400. mu.g/L biotin, 3.4g/L YNB, and 15g/L agar) were applied and incubated at 28 ℃.
4. High copy transformant selection
The transformants in the MD plate were washed with 1mL of sterile water per plate, mixed, plated on YPD plates (composition: 10G/L yeast extract, 20G/L tryptone, 20G/L glucose, 15G/L agar) containing 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6mg/mL and 7mg/mL of G418 (Shanghai Biotechnology), respectively, and cultured at 28 ℃. Plates corresponding to 5mg/mL, 6mg/mL, 7mg/mL G418 yielded 22, 3 and 2 transformants, respectively.
Example 2: shake flask horizontal culture fermentation
Randomly picking transformants in the MD plate of the part 3 in the example 1 and the YPD plate containing 5mg, 6mg and 7mg/mL G418 in the part 4 in the example 1, and transferring the transformants to a test tube containing 2mL of YPD liquid culture medium to culture at 28 ℃ and 230rpm to obtain YPD bacterial liquid; transferring the YPD bacterial liquid to a 20mL shake flask containing 6mL BMGY (composition: 10g/L yeast extract, 20g/L tryptone, 3.4g/LYNB, 100mM potassium phosphate buffer, 400. mu.g/L biotin, 10g/L glycerol) to culture for about 18 hours, and placing the shake flask in a refrigerator at 4 ℃ to freely settle for 6 hours; the supernatant in the flask was carefully decanted, 20mL of BMMY (composition: 10g/L yeast extract, 20g/L tryptone, 3.4g/L YNB, 100mM potassium phosphate buffer, 400. mu.g/L biotin, 0.5% methanol [% by volume of the whole medium ]) was added and the culture was continued, 0.5% methanol was added every 24 hours for induction, and after 96 hours, Tricine-SDS-PAGE was carried out on the culture supernatant, and the high-expression strain was selected as the starting strain for the late-stage study. The optimum methanol addition amount was searched for from the starting strain. Methanol is added at a frequency of 0.5% per 24 hours to 2.0% per 12 hours.
Tricine-SDS-PAGE results show that the transformant of the MD plate has protein expression near 6.5kDa and correct size, as shown in FIG. 2; through gray scale scanning analysis, the strain expression level of the plate corresponding to 5mg/mL G418 is the highest (strip 4, 136mg/L), as shown in FIG. 3, the strain is selected as the starting strain, the strain in the logarithmic growth phase is added with glycerol with the volume fraction of 50% to prepare glycerol strain, and the glycerol strain is placed at-80 ℃ for later use.
For different amounts of methanol added, the results showed that the biomass and the expression were highest (248mg/L) with a volume fraction of 1.0% methanol added every 12 hours, as shown in FIG. 4.
Example 3: horizontal high-density fermentation in 5L fermentation tank
Inoculating the activated glycerol strain into a test tube containing 2mL of YPD liquid culture medium for 6 tubes to obtain a first-stage seed solution;
selecting 4mL of 2 tubes of first-stage seed solution, inoculating the first-stage seed solution into a 500mL shake flask containing 100mL YPD liquid culture medium, culturing for 12 hours at 28 ℃ and 230rpm, and determining that the first-stage seed solution is not infected with infectious microbes through microscopic examination after the culture is finished to be second-stage seed solution;
preparing BSM (40g/L of glycerol, 26.7mL/L of phosphoric acid (85%), 0.93g/L of calcium sulfate dihydrate, 18.2g/L of potassium sulfate, 14.9g/L of magnesium sulfate heptahydrate, 4.13g/L of potassium hydroxide, 4.0mL/L of PTM1 (related components of pichia pastoris microelement 1: 6g/L of copper sulfate pentahydrate, 0.08g/L of sodium iodide, 3g/L of manganese sulfate monohydrate, 0.2g/L of manganese sulfate dihydrate, and sodium molybdate, 0.02g/L of boric acid, 0.5g/L of cobalt chloride, 20g/L of zinc chloride, 65g/L of ferrous sulfate heptahydrate, 0.2g/L of biotin, and 5mL/L of sulfuric acid)), 1/2BSM (40g/L of glycerol, 13.35mL/L of phosphoric acid (85%), 0.465g/L of calcium sulfate dihydrate, 9.1g/L of potassium sulfate, and 5.1 g/L of, 7.45g/L magnesium sulfate heptahydrate, 2.06g/L potassium hydroxide and 4.0mL/L PTM 1.
1/2BSM (40g/L of glycerol, 13.35mL/L of phosphoric acid (85 percent of the volume ratio of the whole culture medium), 0.465g/L of calcium sulfate dihydrate, 9.1g/L of potassium sulfate, 7.45g/L of magnesium sulfate heptahydrate, 2.06g/L of potassium hydroxide, 4.0mL/L of PTM1[ related components of pichia pastoris microelement 1: 6g/L of copper sulfate pentahydrate, 0.08g/L of sodium iodide, 3g/L of manganese sulfate monohydrate, 0.2g/L of dihydrate and sodium molybdate, 0.02g/L of boric acid, 0.5g/L of cobalt chloride, 20g/L of zinc chloride, 65g/L of ferrous sulfate heptahydrate, 0.2g/L of biotin, 5mL/L of sulfuric acid ]).
The fermentation process was the same for both media.
After the pH and the dissolved oxygen electrode are corrected, the culture medium is sterilized along with a 5L fermentation tank;
controlling the temperature at 28 deg.C, adjusting pH to 5.0 with ammonia water, correcting dissolved oxygen by 100%, and pumping the secondary seed solution into fermentation tank via feeding bottle and feeding device. DO (dissolved oxygen level) and Agit (rotational speed) were coupled.
Glycerol feed (50% glycerol [% by mass/volume (W/V) ] +12mL PTM1 (trace element 1)/L) was started after DO rebound and completed at a rate of 15mL/(L × h) for 4 hours.
A rebound of DO was followed by a methanol feed (100% methanol (i.e., neat methanol) +12mL PTM1/L) to set the pH at 5.85 and DO and Agit coupling. Samples were taken every 12 hours for microscopy, wet weight and osmotic pressure.
As a result, when BSM medium was subjected to high-density fermentation, the dissolved oxygen increased sharply at the end of glycerol consumption of 40g/L, and the wet weight of the cells reached 105 g/L. In the glycerol fed-batch fermentation phase, the wet weight reached 180g/L after the end of glycerol consumption. Methanol induction was performed after 1 hour of starvation, and after 132 hours of induction, the wet weight of the cells reached 435g/L and the IP yield reached 3.31g/L, as shown in A, B of FIG. 6.
1/2BSM culture medium, the wet weight of the thallus rapidly increases to 184g/L after the initial 40g/L glycerol feed; in the methanol induction stage, the growth of the cells was slowed down, and reached the maximum wet weight of 519g/L after 156h of induction (i.e., 180h of fermentation), as shown in A of FIG. 6. During the methanol induction phase, IP gradually accumulated with the increase of the induction time, and reached a peak value of 4.51g/L at 132h, as shown in B of FIG. 6.
In addition, the change of the osmotic pressure of the culture medium at different time is detected, the salt ion concentration in the fermentation liquor is continuously reduced due to the absorption of the bacteria by the salt ions, so that the osmotic pressure is reduced along with the prolonging of the culture time, and the osmotic pressure of 1/2BSM culture medium is always lower than that of BSM culture medium, so that the halving of the components of the culture medium promotes the growth and IP expression of the bacteria, and the lack of nutrition is not caused, as shown in C of figure 6. The last 3 times of sampling fermentation supernatant from two fermentations were electrophoretically detected as shown in FIG. 5, and 1/2 the expression level of BSM medium was significantly higher than that of BSM medium.
The invention adopts pPIC9K vector, cloning sites are EcoR I and Not I, and recombinant vector is pPIC 9K-IP. The expression vector is initiated to be transcribed by an AOX1 promoter, and can be strictly regulated and controlled by methanol to carry out secretory expression; the high-density fermentation (5L fermentation tank) adopts the improved BSM yeast basic salt culture medium, has simple and cheap components, and is beneficial to industrial production and purification.
SEQUENCE LISTING
<110> Shanghai institute for pharmaceutical industry, general institute for pharmaceutical industry of China
<120> genetically engineered bacterium for expressing insulin precursor, preparation method and application thereof
<130>P19010009C
<160>1
<170>PatentIn version 3.5
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<211>189
<212>DNA
<213>Artificial Sequence
<220>
<223> insulin precursor
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Claims (10)
1. A genetically engineered bacterium for expressing an insulin precursor is characterized in that the genetically engineered bacterium is an engineered bacterium of an expression vector of the insulin precursor gene integrated in Pichia pastoris (Pichia pastoris), and the nucleotide sequence of the insulin precursor gene is shown as SEQ ID No.1 in a sequence table.
2. The genetically engineered bacterium of claim 1, wherein the pichia pastoris is pichia pastoris GS 115; and/or the skeleton of the expression vector is plasmid pPIC3.5, pPIC9K or pPICZ alpha; and/or, the expression vector contains AOX1 promoter; and/or the expression vector is provided with an anti-geneticin gene, and the genetically engineered bacterium has the performance of resisting geneticin with the concentration of less than 5.0mg/mL, preferably has the performance of resisting the geneticin with the concentration of 0.25-5.0 mg/mL.
3. A gene for coding an insulin precursor is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table.
4. An expression vector having integrated therein the gene encoding insulin precursor according to claim 3;
preferably, the skeleton of the expression vector is plasmid pPIC3.5, pPIC9K or pPICZ alpha; and/or, the expression vector contains AOX1 promoter; and/or the expression vector is provided with an anti-geneticin gene, preferably an anti-geneticin gene with a concentration of less than 5.0mg/mL, more preferably an anti-geneticin gene with a concentration of 0.25-5.0 mg/mL.
5. A method for preparing the genetically engineered bacterium according to claim 1 or 2, comprising the steps of:
(1) constructing the expression vector of claim 4;
(2) transforming the expression vector obtained in the step (1) into pichia pastoris;
(3) and (3) coating the pichia pastoris obtained in the step (2) on a plate containing geneticin for culture, and then selecting transformants on the plate.
6. The method according to claim 5, wherein the conversion in the step (2) is an electrical conversion, and the electrical conversion has a potential of preferably 0.75 to 1.5Kv/cm, more preferably 1.1 Kv/cm;
and/or the concentration of the geneticin in the step (3) is lower than 5.0mg/mL, preferably 0.25-5.0 mg/mL;
and/or, the plate in step (3) is a YPD plate;
and/or the culture condition in the step (3) is 22-30 ℃; preferably 28 deg.c.
7. A method for preparing insulin precursor, which comprises inoculating the genetically engineered bacterium of claim 1 or 2 into a culture medium of Pichia pastoris, and fermenting to obtain insulin precursor from the fermentation broth;
preferably, the fermentation is carried out in a fermentation tank, and/or the culture medium of the pichia pastoris is a BSM culture medium with the concentration of the components except the glycerol and the pichia pastoris trace element 1 reduced to 1/2 at most, preferably 1/2;
more preferably, the reduced-to-1/2 BSM medium includes 4% glycerol, 1.335% phosphoric acid, 0.0465% calcium sulfate dihydrate, 0.91% potassium sulfate, 0.745% magnesium sulfate heptahydrate, 0.206% potassium hydroxide, and 0.4% Pichia pastoris microelement 1; the phosphoric acid and the pichia pastoris microelement 1 are in percentage by volume of each component in the culture medium; the percentage of the other components is the mass volume percentage of each component in the culture medium;
wherein, the pichia pastoris microelement 1 preferably comprises 0.6 percent of blue vitriod, 0.008 percent of sodium iodide, 0.3 percent of manganese sulfate monohydrate, 0.02 percent of dihydrate and sodium molybdate, 0.002 percent of boric acid, 0.05 percent of cobalt chloride, 2 percent of zinc chloride, 6.5 percent of ferrous sulfate heptahydrate, 0.02 percent of biotin and 0.5 percent of sulfuric acid; the percentage of the sulfuric acid is the volume percentage of the sulfuric acid in the pichia pastoris microelement 1; the percentage of the other components is the mass volume percentage of each component in the pichia pastoris microelement 1.
8. The method according to claim 7, wherein methanol is added to the fermentation at a volume fraction of 0.5 to 2.0% every 12 to 24 hours; methanol is preferably added in a volume fraction of 1% every 12 hours.
9. Use of the genetically engineered bacterium of claim 1 or 2, or the gene of claim 3, or the expression vector of claim 4, for the preparation of an insulin precursor.
10. An application of BSM culture medium with the concentration of the rest components reduced to 1/2 except two components of glycerin and pichia pastoris microelement 1 in the preparation of insulin precursor by biological fermentation;
preferably, the BSM medium with the reduced content of 1/2 is the BSM medium with the reduced content of 1/2 described in claim 7, and/or the biological fermentation is fermentation by using the genetically engineered bacterium expressing insulin precursors described in claim 1 or 2.
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