CN111850072B

CN111850072B - Peptibody multi-epitope vaccine fermentation production process and application

Info

Publication number: CN111850072B
Application number: CN201910355952.8A
Authority: CN
Inventors: 邓宁; 张利刚
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-04-29
Filing date: 2019-04-29
Publication date: 2022-08-16
Anticipated expiration: 2039-04-29
Also published as: CN111850072A

Abstract

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, IPTG or lactose is used for inducing fermentation, and various 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.

Description

Peptibody multi-epitope vaccine fermentation production process and application

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 properties and fusing with human IgG1 Fc 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, the IPTG with too high concentration can inhibit the growth of thalli, has potential toxicity to human bodies, is difficult to remove in the production process, and is prohibited to be used as an inducer when recombinant protein for human production is produced in some countries. In addition, when IPTG is induced, target protein is accumulated too fast and 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 economic scale large-scale production of the Peptibody multi-epitope vaccine with low price, high quality and high yield needs to carry out 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 M ₉ The 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 M ₉ A basal medium, which is proliferated under aerobic condition;

(2) feeding: after the thallus enters the logarithmic growth phase, adding M ₉ A 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,

or,

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, the amino acid sequence of the Peptibody multi-epitope vaccine is shown as SEQ ID NO.1, and is recorded in Chinese patent application CN201711202680.5, namely the Peptibody multi-epitope vaccine for inhibiting tumor angiogenesis and application thereof, wherein the vaccine is formed by connecting three bFGF epitope peptides with immunogenicity and three VEGF epitope peptides in series through flexible linker (GGGS) to form multi-epitope peptides, and is coupled with an Fc fragment of IgG 1.

M described in step (1) ₉ The formula of the basic culture medium is preferably as follows: 15.12g/L Na ₂ HPO ₄ ·12H ₂ O，3g/L KH ₂ PO ₄ ，0.5g/L NaCl，1g/L NH ₄ Cl, 20g/L peptone, 0.2g/L MgCl ₂ 0.01% (v/v) glycerol and 10g/L yeast extract; said M ₉ The 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 in the step (2) ₉ The 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) ₉ The addition amount of the feed medium is preferably M ₉ 1/3-3/10 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 is too slow, the rapid proliferation of the thalli is not facilitated, the acetic acid accumulation is caused when the feeding is too fast, and the expression of the recombinant protein is inhibited; the supplementary material is preferably fed in one step 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 IPTG is added in the step (3) for induction, preferably, the IPTG is added at 6 +/-0.5 h after inoculation, and the expression amount of the target protein is highest. Bacterial liquid OD600 _nm Preferably 0.6-0.8 (optionally diluted), OD600 _nm The 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 _nm About 1.0 to 1.2 (optionally diluted), OD600 _nm The 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 rotating speed and the time of proliferation in the step (1) are preferably 220rpm and 4h, the slow growth of thalli can be caused by too low rotating speed and too short time, the too high rotating speed and too long time can cause the too fast growth of thalli, the accumulation of by-product acetic acid and the inhibition of the expression of target proteins; 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 is innovatively designedThe fermentation process concept is adopted, and on the basis of small-amount expression, a 10L fermentation tank and a 100L fermentation tank are further used for mass production of the Peptibody multi-epitope vaccine; 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 ₉ The 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 metabolism and utilization of bacteria, 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 large-scale production of the 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 M ₉ Medium, Lane 2 LB medium, Lane 3M ₉ Medium 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 shaking flask condition optimization using lactose as an inducer, wherein NC-1 is an uninduced Peptibody engineering bacterium, NC-2 is a blank BL21 strain (not containing 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) _nm The 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 the cells and the expression of the target protein, red color is wet weight (g/L), and black color is A600(OD 600) _nm The value of (b), blue is the expression amount (g/L); and the graph C is 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 1-12 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.

Example 1

Stability of target protein expressed by Peptibody engineering bacteria

Referring to Chinese patent application CN201711202680.5, a Peptibody multi-epitope vaccine for inhibiting tumor angiogenesis and the application example 1 thereof, plasmid pET28a-VEGF/bFGF-Fc is constructed and obtained and is transferred into BL21 escherichia coli expression strain, Peptibody engineering bacteria are obtained, the obtained Peptibody engineering bacteria and 50% glycerol are mixed in equal volume, the mixture is preserved at the temperature of 80 ℃, samples are taken every three months for IPTG (final concentration is 0.1mM) induction expression, and SDS-PAGE is used for analyzing the expression conditions of target proteins in the 1 st, the 3 rd, the 6 th, the 9 th and the 12 th months.

Western Blot analysis

(1) Film transfer: two layers of sponge pads, two layers of 3 layers of filter paper, a PVDF membrane activated by methanol and SDS-PAGE glue are respectively overlapped into a membrane transferring device, bubbles in each layer are removed, and membrane transferring is carried out under ice bath conditions (100V and 70 min);

(2) and (3) sealing: washing membrane with PBS-T for 5min, repeating for three times, adding 5% skimmed milk powder prepared with PBS-T, sealing overnight (4 deg.C);

(3) a first antibody: PBS-T was washed three times, and rabbit anti-VEGF-A monoclonal antibody (Abcam, cat # ab52917, 1:1000) was added and incubated for 1h (37 ℃);

(4) secondary antibody: PBS-T membrane washing three times, adding HRP labeled goat anti-rabbit IgG antibody (company: CST, cat #7074, 1:2000) and incubating for 45min (37 ℃);

(5) exposure: washing the membrane with PBS-T for five times, uniformly dropwise adding ECL chemiluminescence solution, and exposing with a gel imager.

The results show that the expression quantities of the proteins of Peptibody engineering bacteria are similar and reach more than 20% within 1-12 months of freezing preservation, and the antigen specificities of the proteins of Peptibody engineering bacteria under various preservation periods have no obvious difference (fig. 2A and B). The result shows that the expression characteristics of the Peptibody engineering bacteria can be kept stable after long-time storage.

Example 2 study of Shake flask culture conditions

1. Screening of the culture Medium

(1) First-stage seed: 5. mu.L of Peptibody-engineered bacteria (1:1000) (1:1000 indicates the ratio of 5. mu.L of Peptibody-engineered bacteria to 5mL of LB medium) + 5. mu.L of 100mg/mL kanamycin solution (1:1000) (1:1000 indicates the ratio of 5. mu.L of 100mg/mL kanamycin solution to 5mL of LB medium, and the final concentration is 100. mu.g/mL) +5mL of LB medium, and culturing overnight (37 ℃, 220 rpm);

(2) secondary seeds: 2mL of primary seed (1:100) +200mL of LB Medium or 200mL of M ₉ Culture medium, culturing for 4h (37 ℃, 220 rpm);

the LB medium (200mL) has the formula as follows: 2g peptone, 2g NaCl and 1g yeast extract;

said M ₉ The formulation of the medium (200mL) was: 3.02g Na ₂ HPO ₄ ·12H ₂ O，0.6g KH ₂ PO ₄ ，0.1g NaCl，0.2g NH ₄ Cl, 4mL volume fraction 50% glycerol, 0.04g MgCl ₂ 4g peptone, and 2g yeast extract; 50% Glycerol and MgCl ₂ Separately sterilizing, sterilizing the rest materials together (121 deg.C, 20min), and supplementing the culture medium before inoculation.

(3) Induction: respectively adding IPTG with working concentration of 0.1mM for 4h induction (28 ℃, 180 rpm);

(4) and (3) collecting thalli: SDS-PAGE was performed to analyze the expression of the target protein.

As a result, the wet weight of the cells in LB medium was 5.75g/L, M ₉ The wet weight of the cells in the medium was 8.5g/L, M ₉ The culture medium can obtain an induction effect similar to that of the LB culture medium, and the expression quantity of the target protein can reach more than 20 percent (figure 3A). The results show that M is selected ₉ The culture medium is used as a fermentation culture medium of Peptibody engineering bacteria, and can improve the wet weight of the bacteria and obtain the high-efficiency expression of target protein.

Optimization of the conditions in the IPTG induced Shake flasks (selection of M for the Medium) ₉ Culture medium)

And (3) researching shaking conditions of factors such as induction temperature, induction concentration, induction time, inoculation quantity, dissolved oxygen, IPTG adding time and the like.

TABLE 1

3. Optimization of lactose-induced Shake flask conditions (Medium M for selection) ₉ Culture medium)

And (3) researching shaking conditions of factors such as induction temperature, induction concentration, induction time, inoculation quantity, dissolved oxygen, lactose adding time and the like.

TABLE 2

The factors such as temperature, inducer concentration, induction time, inoculum size, dissolved oxygen and induction time have important influence on prokaryotic expression, wherein the influence of temperature is the largest, when the temperature is too high, the growth of thalli is too fast, the protein expression speed is too fast, and the factors mainly exist in the form of inclusion bodies, so that the influence on the invention for obtaining soluble target protein is large, therefore, the factors such as temperature, inducer concentration, induction time, inoculum size, dissolved oxygen and induction time are explored in the embodiment, and the optimal target protein expression is obtained.

The results show that, when IPTG is induced (figure 4), the induction temperature with the highest expression quantity of the target protein is 28 ℃, the induction concentration is 0.1mM, the induction time is 4h, the inoculation quantity is 2%, the dissolved oxygen is more than 30%, and the induction time is 6h after inoculation; when lactose is induced (figure 5), the optimal induction temperature with the highest expression level of the target protein is 28 ℃, the induction concentration is 20g/L, the induction time is 12h, the inoculation amount is 8%, the dissolved oxygen amount is more than 30%, and the induction time is 7h after inoculation.

The results show that lactose replacement for IPTG is feasible for inducing expression of proteins of Peptibody engineering order, and fermentation exploration in 10L and 100L tanks is carried out under the optimal shake flask conditions.

4. Distribution of the protein of interest

Respectively preparing thalli induced by IPTG or lactose under the condition of the highest expression level of the target protein.

(1) Ultrasonic crushing: weighing 1.14g of thalli obtained by IPTG or lactose induction, resuspending in 40mL of 20mM PB buffer (phosphate buffer), and carrying out ultrasonication under ice bath conditions (1/8 head breaking, frequency 60Hz, working for 5s, stopping for 5s, and running for 20 min);

(2) collecting supernatant and precipitate: weighing the empty tube, centrifuging for 10min (4000rpm, 4 ℃), weighing the tube, centrifuging for 30min (8000rpm, 4 ℃), weighing the tube, collecting the supernatant and the precipitate, and performing SDS-PAGE analysis;

as a result, the disruption rate of IPTG-induced cells was 97.37%, the disruption rate of lactose-induced cells was 91.23%, and most of the target proteins induced by both were expressed in soluble form (FIG. 3B). The results show that lactose-induced bacteria can be sufficiently disrupted and the target protein can be expressed in a soluble form as compared with IPTG.

Example 3 Peptibody engineering bacteria 10L tank fermentation technology research

The 10L fermenter of this example was made of 10JSA-4, a tank of Shanghai Baoxing Biotechnology engineering Co., Ltd.

(1) The formula of the culture medium is as follows:

5L M ₉ the formulation of the basal medium is shown in table 3.

TABLE 3

Na ₂ HPO ₄ ·12H ₂ O	75.6g
		KH ₂ PO ₄	15g
NaCl	2.5g
		NH ₄ Cl	5g
50% Glycerol	100mL
		MgCl ₂	1g
Peptone	100g
		Yeast extract	50g

1.7L M ₉ The formulation of the feed medium is shown in Table 4.

TABLE 4

Peptone	136g
		Yeast extract	68g
50% Glycerol	680mL
		MgCl ₂	8.5g

50% Glycerol and MgCl ₂ Separately sterilizing, sterilizing the rest materials together (121 deg.C, 20min), and supplementing the culture medium before inoculation.

(2) First-stage seed: 10 μ L Peptibody engineering bacteria (1:1000) +10 μ L kanamycin solution (1:1000) +10mL LB culture medium, overnight culture (37 ℃, 220 rpm);

(3) secondary seeds: 4mL of primary seed (1:100) +400mL of LB medium, cultured for 4h (37 ℃, 220 rpm);

(4) inoculation: the activated secondary seeds were added to M at an inoculum size of 2% (v/v) (i.e., 100mL) or 8% (v/v) (i.e., 400mL), respectively ₉ In a basic culture medium, the temperature is controlled to be 37 ℃, the dissolved oxygen and the rotating speed are connected in series to be more than 40 percent, and the pH value is 7.0;

(5) feeding: 2h after inoculation, a one-time constant-current addition of 1.7L M was carried out ₉ Feeding the culture medium (feeding speed is 10 mL/min);

(6) induction:

IPTG induction (inoculum size 2% (v/v)): reducing the temperature to 28 ℃ in the 6 th hour, controlling the dissolved oxygen to be more than 30%, and adding 0.1mM IPTG (isopropyl thiogalactoside) with the final concentration for induction;

lactose induction (inoculum size 8% (v/v)): reducing the temperature to 28 ℃ in the 7 th hour, controlling the dissolved oxygen to be more than 30%, and adding lactose with the final concentration of 20g/L for induction;

(7) sampling: sampling every 1h, detecting bacterial liquid OD by an ultraviolet spectrophotometer ₆₀₀ The expression quantity of the target protein along with time after SDS-PAGE analysis is carried out;

(8) and (3) discharging: carrying out IPTG continuous induction for 5h, and carrying out lactose continuous induction for 12 h; after the suspension was placed in a tank, the cells were collected by centrifugation at 8000rpm for 10min, and washed 3 times with PB buffer.

When IPTG induction is carried out, the thalli slowly grow (lag phase) 0-2 h after inoculation; feeding with constant current for 2-5 h, wherein the feeding rate is 10mL/min, and the thalli grow rapidly in 2-9 h (logarithmic phase); cooling and reducing dissolved oxygen in the 6 th hour, adding 0.1mM IPTG for induction, and carrying out fluctuation growth of the thalli in the 9 th to 11 th hour (plateau phase); the wet weight of the cells was 92.75g/L at the time of feeding to the tank, and the expression level of the target protein was the highest after 4 hours of induction, and thereafter, the expression level began to decrease (FIGS. 6A and 6B). The result shows that when IPTG is used as an inducer, the thallus growth and target protein expression conditions of the 10L tank Peptibody engineering bacteria fermentation process are consistent with those of a shake flask, the induction time is optimally 4h, and the expression amount of the target protein accounts for more than 20% of the total protein.

When lactose is induced, the inoculated bacteria are in a lag phase for 0-2 h, and the inoculated bacteria grow slowly; in the logarithmic growth phase within 2-7 h, the thalli grow rapidly; in the stage of 7-19 h, the growth of the thalli is stopped; when 20g/L lactose was added at 7h for induction, the cell density increased slowly, SDS-PAGE showed that the expression level of the target protein slowly accumulated with time, the expression level of the target protein at 12h for induction was 20% or more of the total protein, and the wet weight in the lower tank was 99.46g/L (FIG. 6C and FIG. 6D). When IPTG is used for induction, the time when the 2 percent inoculation amount reaches the stage is 9h after inoculation; when lactose is used for induction, the time for 8 percent of the inoculum size to reach the plateau stage is the 7 th hour after inoculation, the inoculum size is improved to 8 percent from 2 percent, and the time for reaching the plateau stage is shortened by 2 hours. The result shows that the lactose induction not only can obtain the induction effect similar to IPTG, but also can improve the wet weight of thalli and the yield of target protein; the time for the thalli to reach the plateau phase can be shortened by increasing the inoculation amount, so that the whole fermentation time is shortened, and the induction time is preferably 12 h.

Example 4 Peptibody engineering bacteria 100L tank fermentation Process

The 100L fermenter used in this example was a jar body from Sartorious Germany, model VP-100 (FIG. 9).

(1) Culture medium formula

50L M ₉ The basal medium is shown in Table 5.

TABLE 5

17L M ₉ The feed medium is shown in Table 6.

TABLE 6

Peptone	1360g
		Yeast extract	680g
50% Glycerol	6.8L
		MgCl ₂	85g

(2) First-stage seed: 50mL of LB medium + 50. mu.L of kanamycin solution (1:1000) + 50. mu.L of Peptibody engineering bacteria (1:1000), culturing overnight at 37 ℃ and 220 rpm;

(3) secondary seeds: culturing 500mL LB culture medium +5mL primary seed (1:100) at 37 deg.C and 220rpm for 4 h;

(4) inoculation: respectively pumping 1L (2% (v/v)) or 4L (8% (v/v)) of secondary seeds into the inoculation pipe, setting the tank pressure at 20mbar, the temperature at 37 ℃, the pH at 7.0 and the dissolved oxygen (the pure oxygen and the rotating speed are connected in series) at more than 40%;

(5) feeding: feeding 17L of feed medium in the 2h after inoculation, wherein the feed rate is 100 mL/min;

(6) induction:

IPTG induction (inoculum size 2% (v/v)): setting the temperature at 28 ℃ in the 6 th hour, and the dissolved oxygen (connected with pure oxygen in series and rotating speed) to be more than 30 percent, and rapidly feeding the final concentration of 0.1mM IPTG;

lactose induction (inoculum size 8% (v/v)): setting the temperature at 28 ℃ in the 7 th hour, the dissolved oxygen (pure oxygen and rotating speed in series) to be more than 30 percent, and rapidly feeding lactose with the final concentration of 20 g/L;

(7) and (3) discharging: after 4h of continuous induction by IPTG, 12h of continuous induction by lactose, centrifuging at 4500rpm for 30min (2L/bottle of centrifuge cup capacity), collecting thalli, and washing with PB buffer solution for 3 times;

(8) sampling: sampling at intervals of 1h, and detecting OD of sample _600nm And wet weight values, SDS-PAGE and software analysis of the expression of the protein of interest.

The inoculation amount induced by IPTG is 2% (v/v), and the thalli slowly grow (adaptation period) in 0-2 h after inoculation; feeding the materials for 2-5 h at a constant flow rate of 100mL/min, and rapidly growing the thalli in 2-9 h (logarithmic phase); cooling and reducing dissolved oxygen in the 6 th hour, adding 0.1mM IPTG for induction, and carrying out fluctuation growth of the thalli in the 9 th to 10 th hour (plateau phase); after induction, the target protein accumulated slowly over time, the wet weight of the cells was 126g/L, the expression level of the target protein was 1.41g/L, and the yield was about 95.88g (FIGS. 7A, B and C). The result shows that when IPTG is used as an inducer, the thallus growth and target protein expression conditions of the 100L tank Peptibody engineering bacteria fermentation process are consistent with those of a 10L tank.

The inoculation amount induced by lactose is 8% (v/v), and the thalli slowly grow (adaptation period) in 0-2 h after inoculation; feeding materials at one time in 2-5 h, wherein the feeding rate is 100mL/min, and the thalli grow rapidly in 2-7 h (logarithmic growth phase); reducing the temperature and dissolved oxygen in the 7 th hour, adding 20g/L lactose for induction, and carrying out fluctuation growth of thalli in the 7 th to 19 th hour (plateau phase); after induction, the target protein accumulated slowly over time, the wet weight of the cells was 137g/L, the expression level of the target protein was 1.39g/L, and the yield was about 105.64g (FIGS. 8A, B and C). The result shows that when lactose is used as an inducer, the thallus growth and target protein expression conditions of the fermentation process of the Peptibody engineering bacteria in the 100L tank are consistent with those of the 10L tank, the lactose induction can realize stable high-density fermentation of the Peptibody engineering bacteria and mass production of Peptibody multi-epitope vaccines, and the wet weight of the thallus and the target protein yield of single fermentation in the 100L tank are higher than those of IPTG induction.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Fermentation production process and application of <120> Peptibody multi-epitope vaccine

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Met Ala Ser Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr Lys Ser Gly

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Gly Gly Gly Ser Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys

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Ser Gly Gly Gly Gly Ser Val Ala Ser Ala Val Phe Tyr Ser Ala Leu

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Val Glu Gly Gly Gly Gly Ser Cys Gly Gly Gly Gly Ser Ala Met Lys

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Glu Asp Gly Arg Leu Leu Ala Ser Lys Gly Gly Gly Gly Ser Ile His

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Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Gly Gly Gly Gly

85 90 95

Ser Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr

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Thr Ser Gly Ser Ser Gly Val Cys Asp Lys Thr His Thr Cys Pro Pro

115 120 125

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

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Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

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Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

165 170 175

Trp Tyr Val Asp Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

180 185 190

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

195 200 205

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

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Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

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Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

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Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

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Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

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Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

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Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

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Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Claims

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

(2) feeding: after the thallus enters the logarithmic growth phase, adding M ₉ A feed medium;

(4) and (3) discharging:

after inducing for 2-12 h, putting the tank down;

the amino acid sequence of the Peptibody multi-epitope vaccine is shown as SEQID NO. 1;

m described in step (1) ₉ The basic culture medium comprises the following components: 15.12g/L Na ₂ HPO ₄ •12H ₂ O，3 g/L KH ₂ PO ₄ ，0.5 g/L NaCl，1 g/L NH ₄ Cl, 20g/L peptone, 0.2g/L MgCl ₂ 0.01% v/v glycerol and 10g/L yeast extract;

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

M described in step (2) ₉ The addition amount of the supplementary culture medium is M ₉ Adding 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 lactose for induction in the step (3), and adding lactose in 7-9 h after inoculation;

the bacterial liquid OD obtained in the step (3) when lactose is added for induction _600nm 1.0 to 1.2;

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 ℃.

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:

the inoculation amount of the seed solution of the Peptibody engineering bacteria in the step (1) is 2-8% 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 ℃.

3. The Peptibody multi-epitope vaccine fermentation production process according to claim 2, 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:

4. The Peptibody multi-epitope vaccine fermentation production process according to claim 2, wherein:

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

the conditions for proliferation described in step (1) were 37 ℃ and pH =7 ± 0.1.

5. 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 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.

6. 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.

7. Use of the Peptibody polyepitope vaccine fermentation production process of any one of claims 1-6 in the production of Peptibody polyepitope vaccines.