阅读说明：本技术 Peptibody多表位疫苗发酵生产工艺与应用 (Peptibody multi-epitope vaccine fermentation production process and application ) 是由 邓宁 张利刚 于 2019-04-29 设计创作，主要内容包括：本发明提供了Peptibody多表位疫苗发酵生产工艺与应用。所述的Peptibody多表位疫苗发酵生产工艺采用了M<Sub>9</Sub>培养基作为发酵培养基,通过IPTG或乳糖诱导发酵,各个环节有机配合实现了Peptibody多表位疫苗的规模生产。其中,所述的乳糖诱导发酵生产工艺能够实现高效稳定的诱导表达,进一步提高湿重和目的蛋白的产量；并可将接种量从2％提高至8％,有效缩短整个发酵时间。本发明成功实现了Peptibody工程菌的高密度发酵和Peptibody多表位疫苗的高效表达,简单稳定、易于放大,100L罐单次发酵生产目的蛋白的产量可达105.64g,具有良好的应用前景和工业生产价值。(The invention provides a Peptibody multi-epitope vaccine fermentation production process and application. The Peptibody multi-epitope vaccine fermentation production process adopts M 9 The culture medium is used as a fermentation medium, and IPTG or lactose is used for inducing fermentation, and all links are organically matched to realize the large-scale production of the Peptibody multi-epitope vaccine. The lactose induced fermentation production process can realize high-efficiency and stable induced expression, and further improves the wet weight and the yield of target protein; and the inoculation amount can be improved from 2% to 8%, and the whole fermentation time is effectively shortened. The invention successfully realizes the high-density fermentation of the Peptibody engineering bacteria and the high-efficiency expression of the Peptibody multi-epitope vaccine, is simple, stable and easy to amplify, has the yield of target protein produced by single fermentation in a 100L tank up to 105.64g, and has good application prospect and industrial production value.)

1. A Peptibody multi-epitope vaccine fermentation production process is characterized by comprising the following steps:

(1) inoculation: inoculating Peptibody engineering bacteria to M₉A basal medium, which is proliferated under aerobic condition;

(2) feeding: after the thallus enters the logarithmic growth phase, adding M₉A feed medium;

(3) induction: under the condition that the dissolved oxygen is lower than that in the step (1),

when the thalli enters the middle logarithmic growth phase, IPTG is added for induction,

alternatively, the first and second electrodes may be,

when the thalli enters a logarithmic growth plateau stage, lactose is added for induction;

(4) and (3) discharging:

when the inducer is IPTG, the mixture is placed in a tank after being induced for 1-6 hours;

when the inducer is lactose, the lactose is induced for 2-12 h and then is taken out of the tank;

the Peptibody multi-epitope vaccine is a Peptibody multi-epitope vaccine for inhibiting tumor angiogenesis, which is disclosed by Chinese patent application with the application number of CN201711202680.5, and the amino acid sequence of the Peptibody multi-epitope vaccine is shown as SEQID NO.1 in Chinese patent application CN 201711202680.5.

2. The Peptibody multi-epitope vaccine fermentation production process according to claim 1, wherein the Peptibody multi-epitope vaccine fermentation production process comprises the following steps:

m described in step (2)₉The addition amount of the supplementary culture medium is M₉Adding 1/3-3/10 of the volume of the basic culture medium;

the final concentration of the lactose in the step (3) is 5-30 g/L;

adding IPTG in the step (3) for induction, and adding IPTG after inoculation in 6 +/-0.5 h;

and (4) adding lactose for induction in the step (3), and adding lactose in the 7 th to 9 th hours after inoculation.

3. The Peptibody multi-epitope vaccine fermentation production process according to claim 1, wherein the Peptibody multi-epitope vaccine fermentation production process comprises the following steps:

m described in step (1)₉The basic culture medium comprises the following components: 15.12g/L Na₂HPO₄·12H₂O，3g/L KH₂PO₄，0.5g/L NaCl，1g/L NH₄Cl, 20g/L peptone, 0.2g/L MgCl₂0.01% (v/v) glycerol and 10g/L yeast extract;

m described in step (2)₉The formula of the supplemented medium is as follows: 80g/L peptone, 40g/L yeast extract, 0.2% (v/v) glycerol and 5g/L MgCl₂；

The bacterial liquid OD600 obtained in the step (3) when lactose is added for induction_nm1.0 to 1.2;

the bacterial liquid OD600 obtained in the step (3) when IPTG is added for induction_nm0.6 to 0.8.

4. The Peptibody multi-epitope vaccine fermentation production process according to claim 1, wherein the Peptibody multi-epitope vaccine fermentation production process comprises the following steps:

the production process also comprises the step of collecting and/or washing thalli after the thalli are put into a tank;

The Peptibody engineering bacteria in the step (1) are seed liquid of the Peptibody engineering bacteria;

feeding the supplementary material in the step (2) in a one-time feeding manner within 2-5 h after inoculation;

the final concentration of the lactose in the step (3) is 15-25 g/L;

the dissolved oxygen in the step (3) is more than 30 percent;

the temperature of induction in the step (3) is 20-40 ℃.

5. The Peptibody multi-epitope vaccine fermentation production process according to claim 4, wherein:

the inoculation amount of the seed liquid of the Peptibody engineering bacteria in the step (1) is 2% (v/v) to 8% (v/v);

the collection is carried out by centrifugation;

the washed thallus is washed by adopting a phosphate buffer solution;

the number of washes is at least 3;

the seed liquid is obtained by culturing in an LB culture medium;

the feeding in the step (2) is one-time fed-batch at the 2h after inoculation;

the final concentration of the lactose in the step (3) is 20 g/L;

the dissolved oxygen in the step (3) is 30-80%;

the temperature of induction in the step (3) is 28-30 ℃.

6. The Peptibody multi-epitope vaccine fermentation production process according to claim 5, wherein:

the centrifugation condition is 4500-8000 rpm for 10-30 min;

The temperature for induction in the step (3) is 28 ℃;

the seed solution is obtained through the following steps:

inoculating the Peptibody engineering bacteria into an LB culture medium containing kanamycin for overnight culture to obtain a first-level seed;

and secondly, inoculating the primary seeds into an LB culture medium to be cultured to obtain secondary seeds.

7. The Peptibody multi-epitope vaccine fermentation production process according to claim 4, wherein:

the inoculation amount of the seed liquid of the Peptibody engineering bacteria in the step (1) is 8% (v/v);

the proliferation conditions in step (1) are 37 ℃ and pH 7 + -0.1;

the final concentration of IPTG described in step (3) was 0.1 mM.

8. The Peptibody multi-epitope vaccine fermentation production process according to claim 1, wherein the Peptibody multi-epitope vaccine fermentation production process comprises the following steps:

the aerobic condition in the step (1) is that the dissolved oxygen is more than 40%;

mixing the Peptibody engineering bacteria in the step (1) with glycerol with the volume fraction of 50% in an equal volume, and storing at-80 ℃;

the feeding speed in the step (2) is 10-100 mL/min;

when the inducer is IPTG, the induction time in the step (4) is 4 h;

when the inducer is lactose, the induction time in the step (4) is 12 h;

The adding mode in the step (2) and/or the step (3) is fed-batch.

9. The Peptibody polyepitope vaccine fermentation process of claim 1, wherein when the Peptibody polyepitope vaccine fermentation is performed in a shake flask:

the rotating speed and the time of proliferation in the step (1) are 220rpm and 4 h;

the temperature and rotational speed of the induction of step (3) were 28 ℃ and 180 rpm.

10. Use of the Peptibody polyepitope vaccine fermentation production process of any one of claims 1-9 in the production of Peptibody polyepitope vaccines.

Technical Field

The invention belongs to the field of microbial genetic engineering, and particularly relates to a Peptibody multi-epitope vaccine fermentation production process and application.

Background

The development of tumor tissues needs blood vessels to provide oxygen and nutrients as well as normal tissues, and tumor blood vessels need to be generated under the combined action of cytokines such as bFGF and VEGF, so that the bFGF and VEGF become molecular targets for effectively inhibiting tumor angiogenesis and tumor cell development. The inventor group obtains three bFGF and three VEGF antigen epitopes by screening through technologies such as bioinformatics, phage display and the like, constructs pET28a recombinant plasmid for improving the pharmaceutical characteristics and fusing with human IgG1Fc fragment genes, and expresses target protein through escherichia coli BL21, namely the Peptibody multi-epitope vaccine.

In the prior research, the applicant also carries out induction expression on recombinant strains, but at present, at least the following problems need to be innovated and solved in order to realize the further breakthrough of the Peptibody multi-epitope vaccine from the research to the production:

(1) the production scale in the laboratory research stage is that the target protein expression is carried out in a container within 1L, and the wet weight of the Peptibody engineering bacteria and the yield of the target protein have large differences from the actual production.

(2) IPTG is a very efficient lactose operon inducer, and a small amount of IPTG can generate a durable induction effect. However, IPTG is expensive, and the large-scale industrial production is limited due to the high induction cost; meanwhile, IPTG at an excessively high concentration can inhibit the growth of thalli, has potential toxicity to human bodies, is difficult to remove in the production process, and is banned from being used as an inducer in the production of recombinant protein for human use in some countries. Moreover, when IPTG is induced, the accumulation of target protein is too fast, and the target protein cannot be folded correctly, so that the generation of inclusion bodies is increased; the IPTG can be added in the middle logarithmic growth phase to induce the thalli effectively and quickly, but the induction time is difficult to grasp, and the yield of the target protein is greatly influenced by slight deviation.

(3) Lactose is non-toxic and low in price, and is used as a substitute of IPTG to induce lactose operon in the prior art. Compared with IPTG, the target protein can be quickly and stably induced in a small amount by diffusion or entering cells by lactose permeating enzyme, and effective and quick induction is generated on thalli; lactose can enter cells only by the transfer of lactose through enzyme, and can play an inducing role only by being converted into isolactose through beta-galactosidase, the affinity and transfer rate of the lactose through the enzyme are not as good as IPTG, and the active transportation and conversion process can occupy the energy of thalli, thereby causing the slow growth of the thalli and the lag of the expression of target protein. The induction process using lactose as an inducer is more complex than IPTG, the lactose induction has the inducer exclusion effect mediated by sugar, the lactose induction is lagged under the condition that carbon sources such as glucose, glycerol and the like exist in a culture medium, and the lactose operon can be started after the carbon sources are exhausted; meanwhile, lactose induction is milder than IPTG, but other metabolic energy of the thalli is sufficient, cell walls are completely synthesized, and the over-hard cell walls are not beneficial to the breaking of the thalli and the release of target protein. Therefore, how to realize the cheap, high-quality and high-yield economic large-scale production of the Peptibody multi-epitope vaccine needs further improvement research on process conditions and the like.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the Peptibody multi-epitope vaccine produced by the prior art, provides a Peptibody multi-epitope vaccine fermentation production process, carries out further innovative research and perfection on the production method of the Peptibody multi-epitope vaccine, amplifies and improves the production process, and adopts M₉The culture medium and the lactose induction technology are innovatively fused, so that the Peptibody multi-epitope vaccine for inhibiting the tumor angiogenesis is produced in a large scale.

The invention also aims to provide the application of the Peptibody multi-epitope vaccine fermentation production method.

The purpose of the invention is realized by the following technical scheme:

a Peptibody multi-epitope vaccine fermentation production process comprises the following steps:

(1) inoculation: inoculating Peptibody engineering bacteria to M₉A basal medium, which is proliferated under aerobic condition;

(2) feeding: after the thallus enters the logarithmic growth phase, adding M₉A feed medium; at the moment, the thalli enter a logarithmic growth phase, the thalli grow rapidly, and the nutrition consumption is fast;

(3) induction: under the condition that the dissolved oxygen is lower than that in the step (1),

when the thalli enters the middle logarithmic growth phase, IPTG is added for induction,

alternatively, the first and second electrodes may be,

When the thalli enters a logarithmic growth plateau stage, lactose is added for induction;

(4) and (3) discharging:

when the inducer is IPTG, the mixture is placed in a tank after being induced for 1-6 hours;

and when the inducer is lactose, the materials are placed in a tank after being induced for 2-12 hours.

The Peptibody multi-epitope vaccine is a novel vaccine designed by the applicant and is described in Chinese patent application CN201711202680.5, the vaccine is formed by connecting three bFGF epitope peptides and three VEGF epitope peptides with immunogenicity in series through flexible linker (GGGS) to form multi-epitope peptides, and the multi-epitope peptides are coupled with an Fc fragment of IgG1, and the amino acid sequence of the multi-epitope peptides is shown as SEQ ID NO.1 in Chinese patent application CN 201711202680.5.

M described in step (1)₉The formula of the basic culture medium is preferably as follows: 15.12g/L Na₂HPO₄·12H₂O，3g/LKH₂PO₄，0.5g/L NaCl，1g/L NH₄Cl, 20g/L peptone, 0.2g/L MgCl₂0.01% (v/v) glycerol and 10g/L yeast extract; said M₉The basic culture medium is added with nutrient components such as inorganic salts, carbon sources and the like, and the wet weight of the Peptibody engineering bacteria and the yield of target protein can be obviously improved.

The Peptibody engineering bacteria in the step (1) are preferably seed liquid of the Peptibody engineering bacteria, and the seed liquid is preferably obtained by culturing in an LB culture medium; more preferably by the following steps:

inoculating the Peptibody engineering bacteria into an LB culture medium containing kanamycin for overnight culture to obtain a first-level seed;

And secondly, inoculating the primary seeds into an LB culture medium to be cultured to obtain secondary seeds.

The overnight culture described in the step (i) is preferably carried out at 37 ℃ and 220 rpm.

The culture conditions in step (II) are preferably at 37 ℃ and 220rpm for 4 h.

The inoculation amount of the seed liquid of the Peptibody engineering bacteria in the step (1) is preferably 2% (v/v) to 8% (v/v); the inoculation amount of the invention can be improved to 8% (v/v), and the plateau phase is reached and the lactose induction is started more quickly, thereby shortening the fermentation time.

In the aerobic condition in the step (1), the dissolved oxygen is preferably over 40 percent, and the low dissolved oxygen is not beneficial to the rapid proliferation of the thalli.

The conditions for the proliferation described in step (1) are preferably 37 ℃ and pH 7 ± 0.1.

The Peptibody engineering bacteria in the step (1) are preferably stored at minus 80 ℃ after being mixed by equal volume of 50% of glycerol, so that the expression level of target proteins of the Peptibody engineering bacteria can be kept to be more than 20% after being stored for 12 months, and the antigen specificity of the target proteins has no obvious difference.

The addition described in step (2) and/or step (3) is preferably fed-batch.

M described in step (2)₉The formulation of the feed medium is preferably: 80g/L peptone, 40g/L yeast extract, 0.2% (v/v) glycerol and 5g/L MgCl ₂。

M described in step (2)₉The addition amount of the feed medium is preferably M ₉1/3-3/10 of the volume of the basic culture medium is added.

The feeding speed in the step (2) is preferably 10-100 mL/min, the feeding speed is too slow, the rapid proliferation of thalli is not facilitated, the acetic acid accumulation can be caused when the feeding speed is too fast, and the expression of recombinant protein is inhibited; the feeding is preferably fed-batch within 2-5 h after inoculation; further preferably, the 2h after the inoculation.

The final concentration of the lactose in the step (3) is preferably 5-30 g/L; further preferably 15 to 25 g/L; most preferably 20 g/L.

The dissolved oxygen in the step (3) is preferably more than 30%; further preferably 30 to 80%; the balance between the dissolved oxygen and the proliferation stage is not favorable for the conversion of the thalli from the proliferation stage to the expression of recombinant protein.

The final concentration of IPTG described in step (3) is preferably 0.1 mM; the expression amount of the target protein is the highest, and the expression of the target protein cannot be increased even inhibited by continuously increasing the IPTG amount.

The inducing temperature in the step (3) is preferably 20-40 ℃; more preferably 28 to 30 ℃ and most preferably 28 ℃.

The induction by adding IPTG in the step (3) is preferably carried out inIPTG is added at 6 +/-0.5 h after inoculation, and the expression quantity of the target protein is highest. Bacterial liquid OD600 _nmPreferably 0.6-0.8 (optionally diluted), OD600_nmThe value of (A) is in the middle section of the A600 curve, the slope is the maximum, the thalli grow rapidly, and the thalli are in the middle stage of logarithmic growth.

Preferably, lactose is added in the 7 th to 9 th hours after inoculation for induction by adding lactose in the step (3), the expression level of the target protein is the highest, and the bacterial liquid OD600 is_nmAbout 1.0 to 1.2 (optionally diluted), OD600_nmThe value of (A) is in the rear section of the A600 curve, the thalli grow slowly, the curve changes in a fluctuation mode, and the thalli are in the later stage of logarithmic growth (plateau stage); the thalli induced by lactose is in a platform stage, the time range is wide, the adding time of an inducer is easier to control, and the operation is easier in industry.

When the inducer is IPTG, the induction time in the step (4) is preferably 4h, and the expression level of the target protein is highest.

When the inducer is lactose, the induction time in step (4) is preferably 12 hours, in which case the expression level of the target protein is highest.

The production process can also comprise a step of collecting and/or washing thalli after the thalli are discharged from the tank.

The collection is preferably carried out by centrifugation, and the centrifugation condition is preferably 4500-8000 rpm for 10-30 min.

The washed cells are preferably washed with a phosphate buffer, and the number of washing is preferably at least 3.

When the Peptibody multi-epitope vaccine fermentation is carried out in a shake flask, the proliferation rotation speed and time in the step (1) are preferably 220rpm and 4h, the slow growth of thalli can be caused by too low rotation speed and too short time, the too high rotation speed and too long time can cause the too fast growth of thalli, the accumulation of byproduct acetic acid and the inhibition of the expression of target protein; the temperature and the rotating speed of the induction in the step (3) are preferably 28 ℃ and 180rpm, under the condition, the target protein is soluble and expressed, the target protein is expressed in an inclusion body due to overhigh temperature, the target protein is inactivated, the rotating speed is continuously maintained at 220rpm, the thallus is inhibited from being converted into the expression stage of the recombinant protein from the proliferation stage, and the temperature is lowered to increase the cost and the instrument condition is difficult to realize due to overlow temperature.

The Peptibody multi-epitope vaccine fermentation production process is applied to the production of the Peptibody multi-epitope vaccine.

The invention innovatively designs a fermentation process concept, and further uses a 10L fermentation tank and a 100L fermentation tank to produce the Peptibody multi-epitope vaccine in a large scale on the basis of small-scale expression; lactose is adopted as an inducer to replace IPTG to induce the expression of the Peptibody multi-epitope vaccine. Firstly, constructing a Peptibody genetic engineering bacteria strain library with target protein stably expressed, and screening out a strain with stable expression characteristics; then optimizing the expression conditions of Peptibody engineering bacteria, and screening out M ₉The culture medium is used as a fermentation culture medium, for lactose induced fermentation, 8% of inoculation amount and dissolved oxygen amount not less than 40% are further preferably used as proliferation conditions, and induction time of 7h after inoculation, induction final concentration of 20g/L lactose, induction temperature of 28 ℃, induction time of 12h and dissolved oxygen amount not less than 30% are more preferably used as expression conditions; and further carrying out a small test on the fermentation process of the Peptibody engineering bacteria in a 10L tank, and carrying out amplification production in a 100L fermentation tank to obtain a final product of the Peptibody engineering bacteria.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a fermentation production scheme with lactose as an inducer, which can efficiently and stably induce the expression of the Peptibody multi-epitope vaccine, and can reduce the induction cost and potential toxicity compared with IPTG; meanwhile, lactose can be used as a carbon source for bacteria to metabolize, so that the wet weight and the yield of the target protein are further improved.

(2) When lactose is induced, the inoculation amount of the invention can be improved from 2% to 8%, and the time of the thalli reaching the plateau stage can be shortened, thereby effectively shortening the whole fermentation time.

(3) The Peptibody multi-epitope vaccine is induced by lactose, has mild conditions, and can promote the correct folding and soluble expression of target protein; the inducer is added in the late logarithmic growth stage of the engineering bacteria, so that the adding time is easy to control, and the yield and quality of the industrially-produced recombinant protein are favorably controlled.

(4) The fermentation production process disclosed by the invention has the advantages that various links are organically matched, the high-density fermentation of Peptibody engineering bacteria and the high-efficiency expression of target protein are realized, the large-scale production in a 100L fermentation tank can be realized, and the optimal working parameters of the 100L fermentation process are optimized, wherein the optimal working parameters comprise the links of proliferation temperature (37 ℃), induction temperature (28 ℃), fermentation pH (7.0), proliferation dissolved oxygen (greater than 40%), induction dissolved oxygen (greater than 30%), feeding rate (100mL/min), induction time (7 h) and induction time (12 h).

(5) The invention provides a fermentation process which is simple, stable and easy to amplify and can produce the Peptibody multi-epitope vaccine in a large scale, the process realizes the high-density fermentation of Peptibody engineering bacteria and the high-efficiency expression of the Peptibody multi-epitope vaccine, and the yield of target protein produced by single fermentation in a 100L tank can reach 105.64 g.

Drawings

FIG. 1 is a flow chart of the Peptibody multi-epitope vaccine fermentation process.

FIG. 2 is a graph showing the results of SDS-PAGE and Western Blot analysis on the preservation stability of Peptibody engineered bacteria; wherein, the picture A is a target protein expression picture, a lane M is a non-prestained protein molecular weight standard, and lanes 1-5 are preserved for 1, 3, 6, 9 and 12 months respectively; FIG. B is a graph showing the results of antigen specificity of the target protein, wherein lanes 1 to 5 are preserved for 1, 3, 6, 9 and 12 months, respectively.

FIG. 3 is a diagram of the result of SDS-PAGE analysis of Peptibody engineering bacteria optimized in shake flask culture conditions; wherein Panel A is a medium screening panel, and lane 1 is M₉Medium, Lane 2 LB medium, Lane 3M₉Medium without inducer, BL21 strain blank (without Peptibody plasmid) in lane 4, and prestained protein molecular weight standard in lane M; panel B is a protein profile of interest, lane M is a non-prestained protein molecular weight standard, lane 1 is an IPTG-induced disrupted supernatant, lane 2 is an IPTG-induced disrupted precipitate, lane 3 is a lactose-induced disrupted precipitate, and lane 4 is a lactose-induced disrupted supernatant.

FIG. 4 is a diagram of the result of SDS-PAGE analysis of IPTG as an inducer under shake flask condition optimization, wherein NC-1 is an uninduced Peptibody engineering bacterium, NC-2 is a blank BL21 strain (without Peptibody plasmid), the induction temperature is 20-36 ℃, the induction concentration is 0.1-0.6 mM, the induction time is 1-6 h, the inoculation amount is 1-8% (v/v), the dissolved oxygen amount is 30-70%, and the induction time is 2-8 h.

FIG. 5 is a diagram of SDS-PAGE analysis result of optimization of shake flask conditions with lactose as inducer, wherein NC-1 is non-induced Peptibody engineering bacteria, NC-2 is blank BL21 strain (without Peptibody plasmid), induction temperature is 24-40 ℃, induction concentration is 5-30 g/L, induction time is 2-12 h, inoculation amount is 2-8% (v/v), dissolved oxygen amount is 30-80%, and induction time is 1-13 h.

FIG. 6 is a graph showing the analysis of the results of 10L tank fermentation of Peptibody engineering bacteria; wherein panels A and C are plots of cell growth; and the target protein expression condition along with time is analyzed by SDS-PAGE, the lane M is a non-prestained protein molecular weight standard, the lanes 1-6 in the B are IPTG induction for 0-5 h, the lanes 1-8 in the D are lactose induction for 0-7 h, and the lane 9 is lactose induction for 12 h.

FIG. 7 is a graph showing the results of a 100L tank fermentation process of Peptibody engineering bacteria under IPTG induction; wherein, graph A is a fermentation tank parameter curve chart, blue is temperature (DEG C), black is dissolved oxygen (%), red is pH, and purple is rotation speed (rpm); FIG. B is a graph showing the growth of cells and the expression of a target protein, red being wet weight (g/L) and black being A600 (i.e., absorbance at 600nm, OD 600)_nmThe value of (b), blue is the expression amount (g/L); and the graph C shows the expression condition of the target protein along with time analyzed by SDS-PAGE, wherein a lane M is a prestained protein molecular weight standard, and lanes 1-5 are IPTG induction for 0-4 h.

FIG. 8 is a graph showing the results of a 100L tank fermentation process of Peptibody engineering bacteria induced by lactose. Wherein, the graph A is a fermentation tank parameter curve chart, blue is temperature (DEG C), black is dissolved oxygen (%), red is pH, and purple is rotation speed (rpm); FIG. B is a graph showing the growth of cells and the expression of a target protein, red being wet weight (g/L) and black being A600(OD 600) _nmThe value of (b), blue is the expression amount (g/L); and the graph C shows the expression of the target protein over time by SDS-PAGE analysis, wherein the lane M is a prestained protein molecular weight standard, and the lanes 1-12 are lactose-induced for 1-12 h.

FIG. 9 is a photograph of a 100L fermenter used in example 4.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.