Fermentation process of gonadotropin-releasing hormone hexamer-diphtheria toxin mutant crm197

Abstract

The application discloses a fermentation process of gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197, and the process comprises the following steps: screening of a fermentation medium and optimization of fermentation conditions. The fermentation medium comprises yeast extract powder, tryptone and NaCl, and the optimization of the fermentation process comprises culture temperature, an inducing agent, concentration of the inducing agent, culture time after induction, adjustment mode of pH of the fermentation liquor, stirring speed, ventilation amount and the like. The fermentation process has the advantages of low production cost, short production cycle (only 12-15 hours are needed), improved expression amount of the target protein per unit of bacteria, and realized high-efficiency expression of the target protein. The GnRH6-CRM197 prepared by the application can be used for immunocastration of various mammals, and has the values of further research and development and good industrialization prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering medicine preparation, in particular to a fermentation process of gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197. BACKGROUND

[0002] Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone releasing hormone (LHRH), gonadotropin-releasing factor (GnRF), is a decapeptide hormone secreted by the hypothalamus of animals, and its main biological function is to control the synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the anterior pituitary cells.

[0003] The reproductive activity of animals is mainly controlled by the hypothalamic-pituitary-gonadal hormones, in which the GnRH secreted by the hypothalamus plays a decisive role in the regulation of reproductive endocrine. When the hypothalamic GnRH neurons are stimulated by some stimulus, GnRH is released along the fibers of the neurons to the median eminence, and enters the anterior pituitary through the portal circulation, directly regulating the synthesis and secretion of pituitary LH and FSH, and LH is the main hormone for the production of steroid hormones such as testosterone (T) and androstenone (A) in the testis. Therefore, blocking or interfering with any part of the hypothalamic-pituitary-gonadal axis can cause the corresponding adjustment of the entire reproductive axis. Some scholars have shown that in mice and other animals, GnRH receptors exist on the ovaries and testes, and GnRH can directly bind to these receptors to inhibit reproductive function. Therefore, by regulating the secretion or interfering with the function of GnRH through appropriate methods, the development of animal reproductive organs and reproductive function can be controlled. Studies have shown that immunizing male animals with GnRH can stop the development of testes in young male animals and cause testicular atrophy in adult male animals. After immunizing animals with GnRH, the body produces a large amount of specific antibodies against GnRH, which binds to endogenous GnRH to lose its biological activity, thereby destroying the function of the hypothalamic-pituitary-testicular axis, inhibiting the release of pituitary LH and FSH, and causing the immunized animals to exhibit testicular atrophy and weight loss, and a decrease in serum T levels. In recent years, some scholars have shown that immunizing dogs with a GnRH vaccine obtained by linking the p3S protein of canine warm virus and the VP6 protein of rotavirus as carrier proteins with the GnRH hexamer gene in series results in testicular atrophy and weight loss in dogs. However, this GnRH hexamer linked to the p3s protein of canine warm virus is only limited to dogs, and its immunization effect needs to be further verified if used in other animals[6]. At present, there have been many reports on GnRH gene engineering vaccines, but none of them has reached commercial application. The reason is that the immunogenicity is not strong and the expression and purification process is complex, resulting in high cost. Therefore, it is necessary to seek new types of GnRH recombinant proteins with simple expression and purification and strong immunogenicity.

[0004] In recent years, domestic and foreign scholars have made certain effects on animals by using GnRH active immunization method to produce antibodies in animals to neutralize endogenous GnRH. This immunization method is simple, safe, can replace traditional surgical castration, control animal sexual behavior and reproductive capacity, improve animal meat quality, promote animal growth and improve feed utilization. At present, synthetic GnRH vaccine adopts chemical synthesis method, that is, GnRH is coupled to some carrier proteins. However, the connection efficiency of GnRH and carrier proteins by chemical synthesis method is low, which increases the cost of producing vaccines and makes the price expensive. In addition, the molecular structure of each batch of hormone antigens synthesized by chemical method is difficult to guarantee to be completely the same, which leads to large differences between batches in commercial production. The production of GnRH vaccine by genetic engineering method can overcome the shortcomings of chemical synthesis method, and the use of operable expression system (such as E. coli expression system) and carrier can produce GnRH vaccine, which has high expression amount and simple extraction process, so that GnRH vaccine can finally be put into the market. Studies have shown that the carrier protein can enhance the immune effect of GnRH, and GnRH and different carrier proteins produce different immune effects. Therefore, it is necessary to select a carrier protein to enhance the immunogenicity of GnRH. Research has proved that diphtheria toxin mutant CRM197 can enhance the immune effect as a carrier protein. Studies have shown that CRM197 has no enzyme activity and toxicity, but has the immunogenicity of diphtheria toxin, and is a good carrier of weak antigen. Therefore, CRM197 is tried to be used as a carrier to improve the immunogenicity of GnRH vaccine. There are many reports about GnRH vaccine, but none of them can be used commercially. The reason is that the immunogenicity is not strong or the cost is high. At present, there is no report on the combination of GnRH hexamer and CRM197 protein, so it is necessary to explore a production method of recombinant protein of GnRH hexamer and CRM197 with high immunogenicity and low cost. SUMMARY

[0005] The purpose of the present application is to provide a fermentation process of gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197, which selects the optimal fermentation medium and fermentation process conditions to achieve high expression of exogenous target protein.

[0006] The fermentation process of gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197 comprises the following steps:

[0007] (1) Preparation of plate strain

[0008] The engineering bacteria BL21(DE3)-pET21a-GnRH6-CRM197 are streak inoculated in an ampicillin-resistant LB culture medium plate and cultured at 37℃ for 12-15h to obtain a plate strain as a original seed.

[0009] (2) Preparation of seed liquid

[0010] The original seed single bacteria are placed in a liquid culture medium to prepare a seed liquid;

[0011] (3) Fermentation culture in a fermentation tank

[0012] The seed liquid is taken and inoculated on a fermentation culture medium in a fermentation tank, and cultured at 37°C for 3h, and then isopropylthio-beta-D galactose is added at a final concentration of 1.0mmol / L, and the culture is continued at 20°C for 12h to obtain the induced expression of the bacterial body;

[0013] (4) Isolation and purification of GnRH6-CRM197

[0014] The induced expression of the bacterial body is centrifuged at 4°C, and the supernatant is discarded, and the induced expression of the bacterial body is resuspended in a Tris buffer solution, and the bacterial liquid is frozen and then thawed on ice water, and the thawed bacterial liquid is ultrasonically broken on an ice water bath, and a protease inhibitor PMSF is added during ultrasonic breaking; the broken bacterial liquid is centrifuged at 4°C, and the precipitate is taken as a crude extract; the crude extract is added to an 8mol / L urea solution and dissolved at 4°C, and after dissolution, it is centrifuged at a temperature of 4°C, and the supernatant is taken, and the supernatant is subjected to His-tag protein purification resin affinity chromatography separation and purification, so as to obtain the target protein GnRH6-CRM197, which is the gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197.

[0015] Preferably, in the step (1), the engineering bacteria BL21(DE3)-pET21a-GnRH6-CRM197 are preserved in the China General Microbiological Culture Collection Center on June 19, 2022, with a preservation number of CGMCC NO. 25027, a classification name of Escherichia coli, a specific name of BL21(DE3)-pET21a-GnRH6-CRM197, and a preservation address of No. 1, Beichen West Road, Beijing City, Chaoyang District, with a postcode of 100101.

[0016] Preferably, in the step (1), the anti-ampicillin LB culture medium plate contains 100mg / mL ampicillin.

[0017] Preferably, in the step (2), the seed liquid is prepared by the following method: the original seed single bacteria are placed in a centrifuge tube containing a liquid culture medium, and the liquid is filled, with a filling coefficient of 0.2, and cultured at a temperature of 37°C and a shaking flask rotation speed of 200rpm for 16h to prepare the seed liquid.

[0018] Preferably, in the step (3), the seed liquid is inoculated on the fermentation medium in the fermenter at an inoculation amount of 1% of the total volume, the liquid loading is 0.2, the temperature is 37℃, the ventilation amount is 8L / min, the rotation speed is 200rpm, the pH value is adjusted to 7.0 by using a Tris buffer solution with a concentration of 50mmol / L, 1.0mmol / L of isopropylthio-beta-D galactose IPTG is added after 3h of culture.

[0019] Preferably, the fermentation medium formula is the same as the liquid medium formula.

[0020] Preferably, the liquid medium comprises the following raw materials in a weight ratio: yeast extract powder 5g / L, tryptone 10g / L, sodium chloride 10g / L, ampicillin 100mg / mL.

[0021] Preferably, in the step (4), the rotation speed of the first centrifugal separation is 4000r / min, the centrifugal time is 40min; the rotation speed of the second centrifugal separation is 10000r / min, the centrifugal time is 20min; the rotation speed of the third centrifugal separation is 5000r / min, the centrifugal time is 10min.

[0022] Preferably, in the step (4), the freezing temperature of the frozen bacteria liquid is to freeze the bacteria liquid at-80℃ or to freeze the bacteria liquid at-20℃ for 24h; the ultrasonic power for ultrasonic crushing is 200W, the crushing time is 20min under the condition of working for 4s and interval for 3s.

[0023] A gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197 prepared according to the fermentation process of the gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197.

[0024] Compared with the prior art, the beneficial effects of the present application are:

[0025] 1) The GnRH6-CRM197 prepared by the present application can be directly applied to animal immunization and castration by mixing with aluminum adjuvant, and compared with other similar preparations on the market, the cost is lower, at the same time, the GnRH6-CRM197 is safe, convenient, and the effect is mild, compared with the surgical castration method, it can reduce infection, stress and other advantages.

[0026] 2) By controlling temperature, induction time, inducer concentration and the like during the culture of the genetically engineered bacteria, the expression amount of the BL21a(DE3)-PET21a-GnRH6-CRM197 produced GnRH6-CRM197 is greatly improved; by using the method of the present application, the expression amount of the target protein GnRH6-CRM197 can reach 30%-50% of the total protein of the bacteria, and the concentration of the GnRH6-CRM197 in the bacteria liquid obtained by fermentation can reach 1.5g / L on average.

[0027] 3) The GnRH6-CRM197 obtained by the present application has a short fermentation time, and only 12-15h is needed from inoculation to the end of fermentation.

[0028] 4) The GnRH6-CRM197 obtained by the present application has a low production cost, and can be produced on a large scale; and the genetically engineered Escherichia coli is easy to be produced industrially, and has a further research value and a good industrialization prospect.

[0029] 5) The GnRH6-CRM197 obtained by the present application has good immunogenicity, can be directly used for animal muscle injection, is simple and easy to operate, and has the effect of controlling the development of animal reproductive organs and reproductive function, and promotes the development of animal castration technology. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is a gel electrophoresis analysis diagram of the recombinant protein GnRH6-CRM197 in the embodiment of the present application, in which 1 refers to a Merker band, 2 refers to a GnRH6-CRM197 band before purification, and 3 and 4 refer to GnRH6-CRM197 bands after purification;

[0031] Figure 2 is a Western blot analysis diagram of the recombinant protein GnRH6-CRM197 after purification in the embodiment of the present application, in which 1 refers to a Mark band, and 2 refers to a GnRH6-CRM197 (71.3KDa) and GnRH antibody reaction band;

[0032] Figure 3 is a testicular diagram of the control group (left) and the test group (right) of male rats in the immunocastration test of rats in the embodiment of the present application;

[0033] Figure 4 is a comparison diagram of testicular weights of the control and test groups in the immunocastration test of rats in the embodiment of the present application

[0034] Figure 5Figure is the test group and control group of the immunological castration test of the rat in the embodiment of the present application, wherein the microscope magnification of figure A and figure C is 100x, the microscope magnification of figure B and figure D is 400x, figure A and figure B are the control group, figure C and figure D are the test group, a is spermatogonium, b is primary spermatocyte, c is spermatid, d is sperm, e is vacuolar degeneration, and f is seminiferous tubule. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0036] The fermentation process of the gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197 includes the following operation steps:

[0037] (1) Preparation of flat plate strain

[0038] The engineering bacteria BL21(DE3)-pET21a-GnRH6-CRM197 are streaked on an LB culture medium containing 100 mg / mL Amp ampicillin, and cultured at a temperature of 37 DEG C for 12-15 hours to obtain a flat plate strain as a primary seed.

[0039] (2) Preparation of seed liquid

[0040] The single colony of the primary seed obtained in (1) is selected and placed in 10 mL of liquid medium in a 50 mL centrifuge tube, and the liquid loading coefficient is 0.2. The seed liquid is prepared by culturing at a temperature of 37 DEG C and a rotation speed of 200 rpm for 16 hours.

[0041] The liquid culture medium includes the following raw materials in a weight ratio: yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and ampicillin 100 mg / mL.

[0042] (3) Fermentation culture in a 10 L fermentation tank

[0043] The fermentation process of the gonadotropin-releasing hormone hexamer-diphtheria toxin mutant CRM197 includes the following operation steps:

[0044] The seed liquid obtained in step (2) is inoculated into 2 L fermentation medium in a 10 L fermenter at an inoculation amount of 1% of the total volume, with a liquid volume of 2 L (liquid volume coefficient of 0.2), at a temperature of 37°C, an aeration amount of 8 L / min, a rotation speed of 200 rpm, and the pH value is adjusted to 7.0 with a Tris buffer having a concentration of 50 mmol / L, and the culture is carried out for 3 h, then 1.0 mmol / L of isopropylthio-β-D galactoside (IPTG) is added, the induction temperature is set to 20°C, and the culture is continued for 12 h, and the fermentation is completed, and the induced expression of the bacterial cells is obtained.

[0045] The fermentation medium formula is the same as the liquid medium formula.

[0046] The induced expression of the bacterial cells obtained is first subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Figure 1 , wherein 1 refers to a Merker band, 2 refers to a GnRH6-CRM197 band before purification, and 3 and 4 refer to GnRH6-CRM197 bands after purification, and the band scanning software photoshop is used to measure the gray value of GnRH6-CRM197, which accounts for 30%-50% of the total protein of the bacterial cells.

[0047] (4) Isolation and purification of GnRH6-CRM197

[0048] The induced expression of the bacterial cells obtained in step (3) is centrifuged at a temperature of 4°C and a rotation speed of 4000 r / min for 40 min, and the supernatant is discarded, and the induced expression of the bacterial cells is resuspended in a column buffer, and the bacterial liquid is frozen at -80°C or frozen at -20°C for 24 h, and then placed on ice water to melt, and then placed in a plastic tube and placed in an ice water bath, and then subjected to ultrasonic cell disruption, and proteinase inhibitor PMSF is added during ultrasonic disruption, and the ultrasonic power is 200 W, and the working time is 4 s, the interval is 3 s, and the working time is 20 min; after ultrasonic disruption, centrifugation is carried out at a temperature of 4°C and a rotation speed of 10000 r / min for 20 min, and the precipitate is taken as the crude extract; the crude extract is added to an 8M urea solution and dissolved at a temperature of 4°C, and then centrifuged at a temperature of 4°C and a rotation speed of 5000 r / min for 10 min, and the supernatant is taken, and the supernatant is subjected to His-tag protein purification resin (His-tag Purification Resin) affinity chromatography separation and purification, and the target protein GnRH6-CRM197 is obtained.

[0049] (5) Evaluation of the reactionogenicity and immunicastration effect of GnRH6-CRM197 produced by fermentation of the gonadotropin-releasing hormone hexamer Escherichia coli-BL21a (GnRH6 E. coli-BL21a):

[0050] A, GnRH6-CRM197 reaction originality identification

[0051] The fermentation produced GnRH6-CRM197 was purified, and Western blot detection showed that it could specifically react with GnRH antibody, and the molecular weight was 71.3 kDa, as shown in Figure 2 . Figure 2 Band 1 represents Mark band, and band 2 represents GnRH6-CRM197 and GnRH antibody reaction band, so it can be seen that the developed GnRH6-CRM197 has good reaction originality with GnRH antibody.

[0052] B, GnRH6-CRM197 immune castration effect monitoring on rats

[0053] 20 five-month-old male rats were randomly divided into two groups: test group and control group, 10 rats in each group. The rats in the test group were subcutaneously injected with purified GnRH6-CRM197 protein vaccine emulsified with aluminum adjuvant on the back in multiple points, 100 μg per rat, and the rats in the control group were injected with aluminum adjuvant. Immunize once every 2 weeks, a total of 3 times. Two months after the end of the third immunization, the testicular tissue was taken, as shown in Figure 3 , the test group and the control group showed obvious difference, the testis of the control group was about 2.5 cm long, and the testis of the test group was about 1.5 cm long, and the testis of the test group showed obvious atrophy. The testis weight statistics of the control group and the test group are shown in Figure 4 , it can be seen that the testis weight in the GnRH6-CRM197 test group decreased significantly. The testicular tissue was observed by histological section, as shown in Figure 5 , the testicular tissue of the rats in the test group showed obvious vacuolar degeneration. The changes of the testicular seminiferous tubules of the rats with vacuolar degeneration were obvious, the spermatogonia, spermatocytes and sperm cells were not distinct, and no sperm was produced. The testicular seminiferous tubules of the rats in the control group were normal, and there were normal spermatogonia, spermatocytes, sperm cells and sperm in the seminiferous tubules, and the levels were distinct, and a large number of sperm were produced. It can be seen that GnRH6-CRM197 inhibits the development of rat testis and eventually atrophy, which shows that the developed GnRH6-CRM197 has strong immunogenicity.

[0054] The above content is only an example and description of the structure of the present application, and those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace, as long as it does not deviate from the structure of the present application or exceed the scope defined by the present claims, which shall belong to the protection scope of the present application.

Claims

1. Fermentation process of Gonadotropin Releasing Hormone hexamer-diphtheria toxin mutant CRM197, characterized in that, The method comprises the following steps: (1) Preparation of flat plate strain The engineering bacteria BL21(DE3)-pET21a-GnRH6-CRM197 is inoculated on an LB culture medium plate containing ampicillin, and is cultured at 37°C for 12-15 hours to obtain a flat plate strain as a primary seed; Among them, the engineered strain BL21(DE3)-pET21a-GnRH6-CRM197 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 19, 2022, with accession number CGMCC NO.25027, and classified as follows: Escherichia coli Specifically named BL21(DE3)-PET21a-GnRH6-CRM197; (2) Preparation of seed liquid The primary seed is placed in a liquid culture medium to prepare a seed liquid; (3) Fermentation culture in a fermentation tank The seed liquid is inoculated in a fermentation culture medium in a fermentation tank, and is cultured at 37°C for 3 hours, and then isopropropylthio-β-D galactoside with a final concentration of 1.0 mmol / L is added, and the culture is continued at 20°C for 12 hours to obtain induced expression bacteria; (4) Separation and purification of GnRH6-CRM197 The induced expression bacteria are centrifuged at 4°C, and the supernatant is discarded, and the induced expression bacteria are resuspended in a Tris buffer solution, and the frozen bacteria liquid is thawed on ice water, and the thawed bacteria liquid is ultrasonically broken on an ice water bath, and a protease inhibitor PMSF is added during the ultrasonic breaking; the broken bacteria liquid is centrifuged at 4°C, and the precipitate is taken as a crude extract; the crude extract is dissolved in a 8 mol / L urea solution at 4°C, and is centrifuged at 4°C after the dissolution, and the supernatant is taken, and the supernatant is subjected to His-tag protein purification resin affinity chromatography separation and purification, so that the target protein GnRH6-CRM197 is obtained, which is the gonadotropin releasing hormone hexamer-diphtheria toxin mutant CRM197.

2. The fermentation process of gonadotropin releasing hormone hexamer-diphtheria toxin mutant CRM197 according to claim 1, characterized in that: In the step (1), the LB culture medium plate contains 100 mg / mL ampicillin.

3. The fermentation process of gonadotropin releasing hormone hexamer-diphtheria toxin mutant CRM197 according to claim 1, characterized in that: In the step (2), the seed liquid is prepared as follows: the primary seed is placed in a centrifugal tube containing a liquid culture medium, and the liquid is filled, and the filling coefficient is 0.2, and the seed liquid is prepared by culturing at 37°C and a shaking flask rotation speed of 200 rpm for 16 hours.

4. The fermentation process of gonadotropin releasing hormone hexamer-diphtheria toxin mutant CRM197 according to claim 1, characterized in that: In the step (3), the seed liquid is inoculated in the fermentation culture medium in the fermentation tank at an inoculation amount of 1% of the total volume, and the liquid is filled, and the filling coefficient is 0.2, and the culture is carried out at 37°C, a ventilation amount of 8 L / min, a rotation speed of 200 rpm, a pH value of 7.0 adjusted by a Tris buffer solution with a concentration of 50 mmol / L, and a final concentration of 1.0 mmol / L of isopropropylthio-β-D galactoside is added after 3 hours of culture.

5. The process for the fermentation of GnRH hexamer-diphtheria toxin mutant CRM197 according to claim 1, characterized in that: The fermentation culture medium formula is the same as the liquid culture medium formula.

6. The fermentation process of gonadotropin releasing hormone hexamer-diphtheria toxin mutant CRM197 according to claim 5, characterized in that: The liquid culture medium comprises the following raw materials in a weight ratio: 5 g / L of yeast extract powder, 10 g / L of tryptone, 10 g / L of sodium chloride, and 100 mg / mL of ampicillin.

7. The process for the fermentation of GnRH hexamer-diphtheria toxin mutant CRM197 according to claim 1, characterized in that: In the step (4), the rotation speed of the first centrifugal separation is 4000 r / min, and the centrifugal time is 40 minutes; the rotation speed of the second centrifugal separation is 10000 r / min, and the centrifugal time is 20 minutes; the rotation speed of the third centrifugal separation is 5000 r / min, and the centrifugal time is 10 minutes. ​ 8. The fermentation process of gonadotropin releasing hormone hexamer-diphtheria toxin mutant CRM197 according to claim 1, characterized in that: In the step (4), the freezing temperature of the frozen bacteria solution is -80℃ or -20℃ for 24 hours; and the ultrasonic power for the ultrasonic crushing is 200W, with 4s working and 3s interval, for 20min.

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