CN105543144A - Culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and fermentation method and application thereof - Google Patents
Culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and fermentation method and application thereof Download PDFInfo
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Abstract
The invention discloses a culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and a fermentation method and application thereof. The fermentation culture medium comprises 10-25 g/L tryptone, 1-20 g/L corn steep liquor, 1-5 g/L KH2PO4, 1-5 g/L K2HPO4, 10-25 g/L yeast extract, 0.1-1.5 g/L (NH4)2SO4, 0.1-1.0 g/L MgSO4.7H2O, 1-15 g/L NaCl, 1-15 ml/L glycerol and the balance of water. The pH value is 6.0-8.0. The culture medium disclosed by the invention is a completely synthesized culture medium, has the advantages of accurate nutrient content and high repeatability, can lower the operation difficulties for large-scale fermentation and after-treatment, greatly reduces the cost for large-scale production, and can obviously enhance the cell quantity and protein expression level.
Description
Technical field
The invention belongs to field of agricultural microbial technology, be specifically related to a kind of be applicable to express crybb2 antigen protein colibacillary substratum and fermentation process and application.
Background technology
Congenital cataract, also known as infant's cataract, is the one of the main reasons of children's blinding, has genetic heterogeneity.The pathogenesis of congenital cataract is also very not clear and definite.Along with the development of molecular genetics, the increasing gene relevant to cataract is found, up to now cloned at least 39 Disease-causing genes, be mainly divided into 4 classes: the various Function protein factors in crystallin gene, gap link protein gene, membrane protein gene and lens growth course.Current investigator thinks, crystallin gene sudden change is the major cause causing hereditary cataract.Crystallin, as structural protein topmost in mammalian lens, maximum feature is stability and the solubility of its height.Crystallin can be divided into for α, β and γ crystallin family, is the important candidate disease causing genes of cataract.Crybb2 genes encoding β B2-crystallin, belong to β/γ crystallin superfamily member, be express maximum albumen in crystallin family, have stronger anti-oxidant and anti-deacylated tRNA amine ability, its function is subject to the adjustment of cell physiological and hereditary property.
Generally, expression amount and the Escherichia coli fermentation density of albumen are closely related, and colibacillary yield is directly connected to the yield of target protein.
Crybb2 genes encoding β B2-crystallin fails secretion to outside born of the same parents as the heterologous protein that this strains tested is expressed, but be present in thalline born of the same parents with the form of secretory product, therefore, for improving recombinant protein yield, reduce volume of culture, promote downstream process efficiency and reduce equipment investment, the present invention is by application high density fermentation technology, large scale culturing is carried out to recombinant escherichia coli strain BL21DE (the 3)/pET28a-crybb2 for examination, study emphatically thalli growth in its fermenting process, substrate consumption, the generation of secondary metabolite acetic acid, running balance and inherent law etc. thereof, determine the damper and the restrictive factor that control fermentating metabolism, realize the regulation and control to its fermentating metabolism process, optimize fermentation medium components, fermentation culture conditions and zymotechnique, and carry out enlarged experiment and industrialization amplification research, realize the high-density high expression level fermentation culture of this strains tested.
Summary of the invention
The object of the present invention is to provide a kind of colibacillary substratum being applicable to express crybb2 antigen protein.This substratum is full-synthetic culture medium, and not only nutrient composition content is accurate, repeatable strong, can reduce the operation easier of large scale fermentation and aftertreatment, and greatly reduce the cost of scale operation.It is crucial that culture medium prescription of the present invention is the special culture media being suitable for producing crybb2 antigen protein, no matter biomass or expressing quantity can be significantly improved.
Present invention also offers a kind of colibacillary fermentation process being applicable to express crybb2 antigen protein, method is simple, easy.
Last object of the present invention there are provided a kind of application being applicable to the colibacillary substratum of expressing crybb2 antigen protein, comprises the industrial fermentation utilizing substratum provided by the invention to carry out recombination bacillus coli.
In order to achieve the above object, the present invention adopts following technical measures:
Be applicable to a colibacillary substratum of expressing crybb2 antigen protein, comprise: Tryptones 10 ~ 25g/L, corn steep liquor 1 ~ 20g/L, KH
2pO
41 ~ 5g/L, K
2hPO
41 ~ 5g/L, yeast extract 10 ~ 25g/L, (NH4)
2sO
40.1 ~ 1.5g/L, MgSO
47H
2o0.1 ~ 1.0g/L, NaCl1 ~ 15g/L, glycerine 1 ~ 15ml/L, surplus is water, and pH value is 6.0 ~ 8.0.
Above-described culture medium prescription, preferred: Tryptones 10 ~ 20g/L, corn steep liquor 5 ~ 10g/L, KH
2pO
41 ~ 3g/L, K
2hPO
42 ~ 5g/L, yeast extract 15 ~ 25g/L, (NH4)
2sO
40.5 ~ 1.5g/L, MgSO
47H
2o0.1 ~ 0.8g/L, NaCl5 ~ 15g/L, glycerine 5 ~ 12ml/L, surplus is water, and pH value is 6.0 ~ 7.5.
Above-described culture medium prescription, preferred: Tryptones 13g/L, corn steep liquor 6g/L, KH
2pO
41.6g/L, K
2hPO
42.2g/L, yeast extract 18g/L, (NH
4)
2sO
41.0g/L, MgSO
47H
2o0.3g/L, NaCl7g/L, glycerine 10ml/L, surplus is water, and pH value is 7.0.
Express a colibacillary fermentation process for crybb2 antigen protein, comprising:
(1) cultivation stage is criticized: the liquid amount of the middle fermention medium of 30L fermentor tank is 5 ~ 25L, the recombination bacillus coli seed liquor being cultured to logarithmic phase is seeded in 30L fermentor tank by 1% ~ 3% (volume ratio) inoculum size, control temperature is 35-38 DEG C, the ammoniacal liquor regulation and control pH value of 25% is added to 6.0-7.5 by stream, continuous adjustment stirring velocity and air flow, control dissolved oxygen 10%, when the glycerine in substratum runs out of full-time, skyrocketing appears in dissolved oxygen, starts to enter the feed-batch culture stage;
The formula of described fermention medium is: Tryptones 10 ~ 25g/L, corn steep liquor 1 ~ 20g/L, KH
2pO
41 ~ 5g/L, K
2hPO
41 ~ 5g/L, yeast extract 10 ~ 25g/L, (NH4)
2sO
40.1 ~ 1.5g/L, MgSO
47H
2o0.1 ~ 1.0g/L, NaCl1 ~ 15g/L, glycerine 1 ~ 15ml/L, surplus is water, and pH value is 6.0 ~ 8.0.
(2) in the feed-batch culture stage: by dissolved oxygen Real-time Feedback, stream adds 75% aseptic glucose solution, and controlling thalline specific growth rate is 0.10-0.15, and controlling dissolved oxygen during feed supplement is 10%.
(3) the abduction delivering stage: as thalline OD in fermented liquid
600add IPTG when being 60 ~ 70, be 0.8 ~ 1.2mM to IPTG final concentration, between inductive phase, control temperature is 15-20 DEG C, and pH value is 6.0 ~ 8.0, and controlling dissolved oxygen is 10%, and induction 12 ~ 18h terminates.
Described recombination bacillus coli is the intestinal bacteria of expressing crybb2 antigen protein.
In the above scheme, preferably, the preparation method expressing the recombination bacillus coli of crybb2 antigen protein comprises:
(1) synthesis of crybb2 gene
The crybb2 gene of synthetic is connected on pGEX-4T carrier and obtains recombinant plasmid pGEX-4T-crybb2.
(2) structure of pET-28a-crybb2 plasmid
Utilize EcoRI+XholI simultaneously enzyme cut pGEX-4T-crybb2 and pET-28a (+), enzyme is cut rear crybb2 gene fragment and cuts rear pET-28a (+) with enzyme and be connected, construct pET-28a-crybb2 plasmid.
(3) transform
Added by pET-28a-crybb2 plasmid in competent cell BL-21 (DE3), spread plate, screens positive strain, namely obtains the recombination bacillus coli of expressing crybb2 antigen protein.
(4) expression identification of goal gene in intestinal bacteria
Recombinant bacterial strain cultivated and collects thalline after carrying out abduction delivering, carrying out sds polyacrylamide gel electrophoresis and westernblot, albumen is identified.
In the above scheme, preferably, identify that the preparation process of the seed liquor of correct recombination bacillus coli comprises:
After being activated on TSA plate by recombination bacillus coli, picking mono-clonal 20mlTSB seed culture medium once increases, and culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 1.0, obtain primary seed solution; Draw 6ml primary seed solution 200mlTSB seed culture medium again and carry out secondary amplification, culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 0.6, obtain secondary seed solution; Then be seeded in fermentor tank by 3% inoculum size.
In the above scheme, preferably, within fermenting process every 10 hours, sample microscopies and measure fermented liquid cell density, weight in wet base and dry weight.Detected the target protein of fermented liquid by SDS-PAGE after fermentation ends.
Compared with prior art, the present invention has the following advantages:
1. compared to other semisynthetic medium or natural medium, the substratum provided of the present invention under the prerequisite considering operation possibility, need be undertaken preparing by the chemical property of its heterogeneity, sterilizing, interpolation, to reach stable utilization of effectively fermenting.
2. the present invention be directed to the high-density high expression level fermentative medium formula designed by recombination bacillus coli producing crybb2 antigen protein in born of the same parents, culture medium prescription provided by the invention can ensure that strains tested is normal at certain specific growth rate situation hypothallus growth metabolism, obtain higher biomass, the expression amount of crybb2 albumen can be made again to be significantly improved, the requirement of scale operation can be met completely.
3. utilize substratum provided by the invention and fermentation process, substratum of the present invention can reach 210g/L at fermentation termination biomass, the expressing quantity of often liter of fermented liquid can reach 17.6g, improve about 3 times than conventional commercial medium TSB culture medium protein expression amount, and fermentative production cost reduces nearly 60%.
Accompanying drawing explanation
Fig. 1 crybb2 gene PCR amplification figure.
Swimming lane 1 and 2 is crybb2 gene amplification figure.
Fig. 2 is pET-28a-crybb2 plasmid map schematic diagram in the present invention.
Fig. 3 is recombinant antigen SDS-PAGE electrophoresis in the present invention.
Swimming lane: 1 is marker; 2 is empty plasmid PGEX-4T; 3 do not induce for recombinant bacterium (BL21DE (3)/pET28a-crybb2); 4 induce 1h for recombinant bacterium (BL21DE (3)/pET28a-crybb2); 5 induce 2h albumen for recombinant bacterium (BL21DE (3)/pET28a-crybb2); 6 for induce 3h albumen for recombinant bacterium (BL21DE (3)/pET28a-crybb2);
Fig. 4 is the thalli growth curve synoptic diagram of recombination bacillus coli BL21DE (3)/pET28a-crybb2 in 30L fermentor tank producing crybb2 albumen in embodiment 5.
Embodiment
Embodiment of the present invention technical scheme used, as special standby explanation, is the conventional scheme of this area; Described reagent or material, if not otherwise specified, all derive from commercial channel.
Embodiment 1:
Express the structure of recombination bacillus coli BL21DE (the 3)/pET28a-crybb2 of crybb2 antigen protein:
(1) synthesis of crybb2 gene
Crybb2 gene (Genebank:CR456426.1) is synthesized in Shanghai Sheng Gong biotechnology company limited, altogether 615bp (shown in SEQIDNO.1) (Fig. 1), and be connected on pGEX-4T carrier and obtain recombinant plasmid pGEX-4T-crybb2.
(2) structure of pET-28a-crybb2 plasmid
Utilize EcoRI+XholI (purchased from precious biological (Dalian) company limited product) simultaneously enzyme cut crybb2 gene fragment and the pET-28a (+) of pGEX-4T-crybb2, enzyme is cut rear crybb2 gene fragment to cut rear pET-28a (+) with enzyme and be connected (Fig. 2), construct pET-28a-crybb2 plasmid.
(3) transform
Getting competent cell BL-21 (DE3) 100 μ l joins in 1.5mlEP pipe, and pET-28a-crybb2 plasmid 0.5 μ l is added and mixed.Put on ice after 30min, 42 DEG C of heat shocks 90 seconds, ice bath 3min-5min.Coated the LB agar plate containing 25 μ g/mlKna.Just put 1h, then flat board is turned over for 37 DEG C, be inverted 37 DEG C of cultivation 14h ~ 16h and occur to bacterium colony.The bacterium colony obtained is recombination bacillus coli BL21DE (the 3)/pET28a-crybb2 expressing crybb2 antigen protein.
(4) expression identification of goal gene in intestinal bacteria
Recombination bacillus coli BL21DE (3)/pET28a-crybb2 is inoculated in the 3mLLB liquid nutrient medium containing 25 μ g/mlKna, cultivates in 37 DEG C of shaking tables.From cultured bacterium liquid, get 100 μ L be inoculated in 10mL and contain in the fresh LB liquid nutrient medium of 25 μ g/mlKna, be about 3h in 37 DEG C of shaking culture, when reaching 0.6 to OD600, adding IPTG to final concentration is 1.2mmol/L, and 18 DEG C are continued to cultivate, collect thalline after 3h.And carry out sds polyacrylamide gel electrophoresis (Fig. 3) and westernblot identifies albumen, qualification result shows, the recombination bacillus coli prepared of the present embodiment can correction crybb2 albumen, as the strains tested of following large-scale fermentation culture.
Embodiment 2:
Express a colibacillary fermentation process for crybb2 antigen protein, comprising:
A. the preparation of fermention medium: Tryptones 500g, corn steep liquor 400g, KH
2pO
4100g, K
2hPO
4100g, yeast extract 500g, (NH
4)
2sO
430g, MgSO
47H
2o20g, NaCl300g, glycerine 300ml, is dissolved in 20L distilled water, and adjusted to ph is 7.5, and be transferred in 30L fermentor tank, under 121 DEG C of high pressure steam, sterilizing 30min, is fermention medium after cooling.
B. the preparation of feed-batch culture stage glucose: take glucose 150g, and dissolve with distilled water and be settled to 200ml, under 115 DEG C of high pressure steam, sterilizing 25min is for subsequent use as 75% glucose after cooling.The easy carbonization of glucose when sterilising temp is more than 115 DEG C, produces furfural, unfavorable to the growth of thalline.
The preparation of fermentation seed liquid: after being activated on TSA plate by recombination bacillus coli BL21DE (3)/pET28a-crybb2, picking mono-clonal 20mlTSB seed culture medium once increases, and culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 1.0, obtain primary seed solution; Draw 6ml primary seed solution 200mlTSB seed culture medium again and carry out secondary amplification, culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 0.6, obtain fermentation seed liquid;
C. fermentation processes:
(1) criticize cultivation stage: the inoculum size of fermentation seed liquid is 3%, and leavening temperature controls 37 DEG C, add the ammoniacal liquor regulation and control pH value of 25% 7.5 by stream, constantly regulate stirring velocity and air flow, control dissolved oxygen 10%.When the glycerine in fermention medium runs out of full-time, dissolved oxygen there will be and skyrockets, and now fermentation starts to enter the feed-batch culture stage.
(2) in the feed-batch culture stage: by dissolved oxygen Real-time Feedback, carry out 75% aseptic glucose solution of exponential fed-batch when control thalline specific growth rate is 0.10, during feed supplement, dissolved oxygen controls 10%.
(3) the abduction delivering stage: as fermented liquid OD
600start when being 62 to carry out isopropylthiogalactoside (IPTG) induction, the final concentration to IPTG is 1.2mM, temperature is controlled at 18 DEG C between inductive phase, and pH value is 7.5, and dissolved oxygen still controls 10%, terminates after induction 15h.
Ferment to strains tested by aforesaid operations, result is: earlier fermentation thalli growth is vigorous, the OD when fermenting 30h
600reach 62, induction 15h terminates fermentation, and fermentation termination biomass is that the expression amount of 180g/L, crybb2 albumen reaches 14.6g/L.
Embodiment 3:
Express a colibacillary fermentation process for crybb2 antigen protein, comprising:
A. the preparation of fermention medium: Tryptones 200g, corn steep liquor 200g, KH
2pO
420g, K
2hPO
440g, yeast extract 300g, (NH
4)
2sO
410g, MgSO
47H
2o2g, NaCl100g, glycerine 100ml, is dissolved in 20L distilled water, and adjusted to ph is 6.5, is transferred in 30L fermentor tank, sterilizing 30min under 121 DEG C of high pressure steam, has both been fermention medium after cooling.
B. the preparation of feed-batch culture stage glucose: take glucose 150g, and dissolve with distilled water and be settled to 200ml, under 115 DEG C of high pressure steam, sterilizing 25min is for subsequent use as 75% glucose after cooling.The easy carbonization of glucose when sterilising temp is more than 115 DEG C, produces furfural, unfavorable to the growth of thalline.
The preparation of fermentation seed liquid: after being activated on TSA plate by recombination bacillus coli BL21DE (3)/pET28a-crybb2, picking mono-clonal 20mlTSB seed culture medium once increases, and culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 1.0, obtain primary seed solution; Draw 6ml primary seed solution 200mlTSB seed culture medium again and carry out secondary amplification, culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 0.6, obtain fermentation seed liquid;
C. fermentation processes:
(1) criticize cultivation stage: the inoculum size of fermentation seed liquid is 3%, and leavening temperature controls 37 DEG C, add the ammoniacal liquor regulation and control pH value of 25% 6.0 by stream, constantly regulate stirring velocity and air flow, control dissolved oxygen 10%.When the glycerine in fermention medium runs out of full-time, dissolved oxygen there will be and skyrockets, and now fermentation starts to enter the feed-batch culture stage.
(2) in the feed-batch culture stage: by dissolved oxygen Real-time Feedback, carry out 75% aseptic glucose solution of exponential fed-batch when control thalline specific growth rate is 0.10, during feed supplement, dissolved oxygen controls 10%.
(3) the abduction delivering stage: as fermented liquid OD
600start when being 60 to carry out isopropylthiogalactoside (IPTG) induction, the final concentration to IPTG is 1.2mM, temperature is controlled at 18 DEG C between inductive phase, and pH value is 7.0, and dissolved oxygen still controls 10%, terminates after induction 15h.
Ferment to strains tested by aforesaid operations, result is: earlier fermentation thalli growth is vigorous, the OD when fermenting 30h
600reach 60, induction 15h terminates fermentation, and fermentation termination biomass is that the abduction delivering amount of 172g/L, crybb2 albumen reaches 13.8g/L.
Embodiment 4:
Express a colibacillary fermentation process for crybb2 antigen protein, comprising:
A. the preparation of fermention medium: Tryptones 260g, corn steep liquor 120g, KH
2pO
432g, K
2hPO
444g, yeast extract 360g, (NH4)
2sO
420g, MgSO
47H
2o6g, NaCl140g, glycerine 200ml, is dissolved in 20L distilled water, and adjusted to ph is 7.0, is transferred in 30L fermentor tank, sterilizing 30min under 121 DEG C of high pressure steam, has both been fermention medium after cooling.
B. the preparation of feed-batch culture stage glucose: take glucose 150g, and dissolve with distilled water and be settled to 200ml, under 115 DEG C of high pressure steam, sterilizing 25min is for subsequent use as 75% glucose after cooling.The easy carbonization of glucose when sterilising temp is more than 115 DEG C, produces furfural, unfavorable to the growth of thalline.
The preparation of fermentation seed liquid: after being activated on TSA plate by recombination bacillus coli BL21DE (3)/pET28a-crybb2, picking mono-clonal 20mlTSB seed culture medium once increases, and culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 1.0, obtain primary seed solution; Draw 6ml primary seed solution 200mlTSB seed culture medium again and carry out secondary amplification, culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 0.6, obtain fermentation seed liquid;
C. fermentation processes:
(1) criticize cultivation stage: the inoculum size of fermentation seed liquid is 3%, and leavening temperature controls 37 DEG C, adding the ammoniacal liquor regulation and control pH value of 25% 7.0 by stream, by regulating stirring velocity and air flow, controlling dissolved oxygen 10%.When the glycerine in fermention medium runs out of full-time, dissolved oxygen there will be and skyrockets, and now fermentation starts to enter the feed-batch culture stage.
(2) in the feed-batch culture stage: by dissolved oxygen Real-time Feedback, carry out 75% aseptic glucose solution of exponential fed-batch when control thalline specific growth rate is 0.10, during feed supplement, dissolved oxygen controls 10%.
(3) the abduction delivering stage: as fermented liquid OD
600start when being 70 to carry out isopropylthiogalactoside (IPTG) induction, the final concentration to IPTG is 1.2mM, temperature is controlled at 18 DEG C between inductive phase, and pH value is 6.0, and dissolved oxygen still controls 10%, terminates fermentation after induction 15h.
Ferment to strains tested by aforesaid operations, result is: earlier fermentation thalli growth is vigorous, the OD when fermenting 30h
600reach 70, induction 15h terminates fermentation, and fermentation termination biomass can reach 210g/L, and the abduction delivering amount of crybb2 albumen reaches 17.6g/L.Fermentation diagram as shown in Figure 4.
Embodiment 5:TSB substratum fermenting experiment
The preparation of A.TSB fermention medium: take 600gTSB powder, be settled to 20L with single water that steams, being transferred in 30L fermentor tank, sterilizing 30min under 121 DEG C of high pressure steam, had both been fermention medium after cooling.
B. the preparation of feed-batch culture stage glucose: take glucose 150g, and dissolve with distilled water and be settled to 200ml, under 115 DEG C of high pressure steam, sterilizing 25min is for subsequent use as 75% glucose after cooling.The easy carbonization of glucose when sterilising temp is more than 115 DEG C, produces furfural, unfavorable to the growth of thalline.
The preparation of fermentation seed liquid: after being activated on TSA plate by recombination bacillus coli BL21DE (3)/pET28a-crybb2, picking mono-clonal 20mlTSB seed culture medium once increases, and culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 1.0, obtain primary seed solution; Draw 6ml primary seed solution 200mlTSB seed culture medium again and carry out secondary amplification, culture condition is 37 DEG C, and 200r/min, is cultured to OD
600be 0.6, obtain fermentation seed liquid;
C. fermentation processes:
(1) criticize cultivation stage: the inoculum size of fermentation seed liquid is 3%, and leavening temperature controls 37 DEG C, add the ammoniacal liquor regulation and control pH value of 25% 7.0 by stream, constantly regulate stirring velocity and air flow, control dissolved oxygen 10%.When the glycerine in fermention medium runs out of full-time, dissolved oxygen there will be and skyrockets, and now fermentation starts to enter the feed-batch culture stage.
(2) in the feed-batch culture stage: by dissolved oxygen Real-time Feedback, carry out 75% aseptic glucose solution of exponential fed-batch when control thalline specific growth rate is 0.10, during feed supplement, dissolved oxygen controls 10%.
(3) the abduction delivering stage: as fermented liquid OD
600when being 3.8, start to carry out isopropylthiogalactoside (IPTG) induction, the final concentration of IPTG is 1.2mM, temperature is controlled at 18 DEG C between inductive phase, and pH value is 6.0, and dissolved oxygen still controls 10%, terminates fermentation after induction 15h.
By TSB, recombination bacillus coli is expressed, the OD when fermenting 6h
600reach 3.8, namely start induction, temperature controlled at 18 DEG C between inductive phase, induction 15h terminates fermentation, and the expressing quantity after induction terminates is 5.8g/L.
SEQUENCELISTING
Ai Bo Tyke, <110> Wuhan bio tech ltd
<120> mono-kind is applicable to express the colibacillary substratum of crybb2 antigen protein and fermentation process and application
<130> mono-kind is applicable to express the colibacillary substratum of crybb2 antigen protein and fermentation process and application
<160>1
<170>PatentInversion3.1
<210>1
<211>615
<212>DNA
<213> artificial sequence
<400>1
atggcctcagatcaccagacccaggcgggcaagccacagtccctcaaccccaagatcatc60
atctttgagcaggaaaactttcaaggccactcgcatgagctcaatgggccctgccccaac120
ctgaaggaaactggcgtggagaaggcaggttctgtcctagtgcaggctggaccctgggtg180
ggctatgaacaggccaactgcaagggcgagcagtttgtgtttgagaagggtgagtacccc240
cgctgggactcatggaccagcagccgaaggacggactccctcagctccctgaggcccatc300
aaagtggacagccaagagcacaagatcatcctctatgaaaaccccaacttcaccgggaag360
aagatggaaatcatagatgacgatgtacccagcttccacgcccatggctaccaggagaag420
gtgtcatctgtgcgggtgcagagtggcacgtgggttggctaccagtaccccggctaccgt480
ggactgcagtacctgctggagaagggagactacaaggacagcagcgactttggggcccct540
cacccccaggtgcagtccgtgcgccgtatccgcgacatgcagtggcaccaacgtggtgcc600
ttccacccctccaac615
Claims (6)
1. be applicable to a colibacillary substratum of expressing crybb2 antigen protein, comprise: Tryptones 10 ~ 25g/L, corn steep liquor 1 ~ 20g/L, KH
2pO
41 ~ 5g/L, K
2hPO
41 ~ 5g/L, yeast extract 10 ~ 25g/L, (NH4)
2sO
40.1 ~ 1.5g/L, MgSO
47H
2o0.1 ~ 1.0g/L, NaCl1 ~ 15g/L, glycerine 1 ~ 15ml/L, surplus is water, and pH value is 6.0 ~ 8.0.
2. substratum according to claim 1, is characterized in that: Tryptones 10 ~ 20g/L, corn steep liquor 5 ~ 10g/L, KH
2pO
41 ~ 3g/L, K
2hPO
42 ~ 5g/L, yeast extract 15 ~ 25g/L, (NH4)
2sO
40.5 ~ 1.5g/L, MgSO
47H
2o0.1 ~ 0.8g/L, NaCl5 ~ 15g/L, glycerine 5 ~ 12ml/L, surplus is water, and pH value is 6.0 ~ 7.5.
3. substratum according to claim 1, is characterized in that: Tryptones 13g/L, corn steep liquor 6g/L, KH
2pO
41.6g/L, K
2hPO
42.2g/L, yeast extract 18g/L, (NH
4)
2sO
41.0g/L, MgSO
47H
2o0.3g/L, NaCl7g/L, glycerine 10ml/L, surplus is water, and pH value is 7.0.
4. express a colibacillary fermentation process for crybb2 antigen protein, comprising:
(1) cultivation stage is criticized: the liquid amount of the middle fermention medium of 30L fermentor tank is 5 ~ 25L, the recombination bacillus coli seed liquor being cultured to logarithmic phase is seeded in 30L fermentor tank by 1% ~ 3% inoculum size, control temperature is 35-38 DEG C, the ammoniacal liquor regulation and control pH value of 25% is added to 6.0-7.5 by stream, continuous adjustment stirring velocity and air flow, control dissolved oxygen 10%;
The formula of described fermention medium is: Tryptones 10 ~ 25g/L, corn steep liquor 1 ~ 20g/L, KH
2pO
41 ~ 5g/L, K
2hPO
41 ~ 5g/L, yeast extract 10 ~ 25g/L, (NH4)
2sO
40.1 ~ 1.5g/L, MgSO
47H
2o0.1 ~ 1.0g/L, NaCl1 ~ 15g/L, glycerine 1 ~ 15ml/L, surplus is water, and pH value is 6.0 ~ 8.0;
(2) in the feed-batch culture stage: by dissolved oxygen Real-time Feedback, stream adds 75% aseptic glucose solution, and controlling thalline specific growth rate is 0.10-0.15, and controlling dissolved oxygen during feed supplement is 10%;
(3) the abduction delivering stage: as thalline OD in fermented liquid
600add IPTG when being 60 ~ 70, be 0.8 ~ 1.2mM to IPTG final concentration, between inductive phase, control temperature is 15-20 DEG C, and pH value is 6.0 ~ 8.0, and controlling dissolved oxygen is 10%, and induction 12 ~ 18h terminates;
Described recombination bacillus coli is the intestinal bacteria of expressing crybb2 antigen protein.
5. method according to claim 4, the preparation method of the recombination bacillus coli of described expression crybb2 antigen protein comprises:
(1) synthesis of crybb2 gene
The crybb2 gene of synthetic is connected on pGEX-4T carrier and obtains recombinant plasmid pGEX-4T-crybb2;
(2) structure of pET-28a-crybb2 plasmid
Utilize EcoRI+XholI simultaneously enzyme cut pGEX-4T-crybb2 and pET-28a (+), enzyme is cut rear crybb2 gene fragment and cuts rear pET-28a (+) with enzyme and be connected, construct pET-28a-crybb2 plasmid;
(3) transform
PET-28a-crybb2 plasmid is added competent cell BL-21(DE3) in, spread plate, screens positive strain, namely obtains the recombination bacillus coli of expressing crybb2 antigen protein.
6. the application of substratum according to claim 1 in recombination bacillus coli industrial fermentation; Described recombination bacillus coli is the intestinal bacteria of expressing crybb2 antigen protein.
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