CN114806879A - Inactivation method of high-concentration bacterial ghost vaccine - Google Patents

Abstract

The invention relates to a method for inactivating a high-concentration ghost vaccine, which specifically comprises the following steps: a) after the bacterial culture is finished, raising the temperature of the tank body, and performing inactivation culture; b) replacing the fermentation tank at intervals of 8-72 h. The bacterial liquid obtained by the method provided by the invention completely does not contain live bacteria, the treated bacterial strain has good immunocompetence, and the bacterial strain can play a role in completely removing the live bacteria in high-concentration bacterial liquid, and can be applied to large-scale industrial production of vaccines.

Description

Inactivation method of high-concentration bacterial ghost vaccine

Technical Field

The invention relates to the technical field of biology, in particular to a method for inactivating bacterial ghost.

Background

Bacterial Ghost (BGs) is a Bacterial body without cytoplasm and nucleic acid, is a Bacterial hollow shell with a complete Bacterial structure formed after gram-negative bacteria are cracked, and has no protein denaturation in the preparation process, so that the Bacterial ghost keeps a Bacterial cell membrane structure and related antigen protein which are completely the same as those of live bacteria, thereby being capable of keeping immunogenicity similar to that of the live bacteria, and simultaneously stimulating an organism to generate humoral immunity and cellular immunity, and the cross protection capability is superior to that of an inactivated vaccine. Meanwhile, BGs do not contain genetic materials and only contain bacterial empty shells, so the possibility of returning toxicity and recovering toxicity does not exist, toxic and side effects are not caused, and the safety is far better than that of live vaccines.

At present, the most main preparation method of the bacterial ghost is a genetic engineering method, namely, a pore channel is formed on a bacterial cell membrane through a bacterial lytic gene (such as a lytic gene E of bacteriophage phiX 174), so that the content of the bacterial cell is discharged to form the bacterial ghost, and then, an inactivating agent is used for secondary inactivation. However, too high a concentration of the inactivating agent may damage the bacterial ghost and affect the immunogenicity of the bacterial ghost, and too low a concentration of the inactivating agent may cause the bacterial pathogen not to be completely inactivated. In addition, the inactivation may not be complete due to decomposition, chemical reaction and the like of the inactivator, and decomposition products exist, so that the vaccine immunization risk is increased.

Because the influence of the bacterial residues on the safety of the vaccine is great, especially in the vaccine product with the brucella zoonosis, the incomplete inactivation of the bacteria has great potential safety hazard to both animals and human beings. Therefore, complete inactivation of live bacteria in the ghost vaccine, particularly complete inactivation of live bacteria in a high-concentration ghost vaccine, while maintaining immunogenicity and ghost structure, is one of the difficulties in the ghost preparation technology in the prior art, and needs to be solved urgently. In view of this, the present invention particularly provides a method for inactivating a high-concentration ghost vaccine.

Disclosure of Invention

The invention aims to provide a method for inactivating a high-concentration ghost vaccine, which is used for solving the problems that live bacteria in the preparation process of the high-concentration ghost vaccine are completely inactivated and the complete structure and immunogenicity of bacteria are kept.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a method for inactivating a high-concentration ghost vaccine comprises the following steps:

a) after the bacterial culture is finished, raising the temperature of the tank body, and performing inactivation culture;

b) replacing the fermentation tank at intervals of 8-72 h.

In the inactivation method of the high-concentration bacterial ghost vaccine, the temperature of the tank body in the step a) is raised to 42 ℃, and the culture is carried out at 100 r/min.

In the inactivation method of the high-concentration ghost vaccine, the operation step of replacing the fermentation tank at one time in the step b) is to collect all fermentation liquor in the fermentation tank and then pump the fermentation liquor into another sterile fermentation tank.

In the inactivation method of the high-concentration ghost vaccine, the tank-pouring interval time in the step b) is preferably 12h-36h, and more preferably 24 h.

In the inactivation method of the high-concentration ghost vaccine, the time interval between the tank inversions in the step b) can be 8h, 12h, 24h, 36h and 72h, and preferably 24 h.

Further, the total time of the antigen inactivation culture in the inactivation method of the high-concentration ghost vaccine is at least 3 days, namely 72 hours.

Limited by the effectiveness of current high concentration culture techniques, in some embodiments of the invention, the inactivation treatment is performed at a concentration of 4.5X 10 ¹⁰ CFU/ml、5.0×10 ¹⁰ CFU/ml、5.5×10 ¹⁰ CFU/ml、6.0×10 ¹⁰ CFU/ml、6.5×10 ¹⁰ CFU/ml、7.0×10 ¹⁰ CFU/ml、7.5×10 ¹⁰ CFU/ml、8.0×10 ¹⁰ CFU/ml、8.5×10 ¹⁰ CFU/ml、9.0×10 ¹⁰ CFU/ml、9.5×10 ¹⁰ CFU/ml、10 ¹¹ CFU/ml。

In some embodiments of the invention, the concentration of the inactivated bacteria solution is not less than 4.5X 10 ¹⁰ CFU/ml, with the development of high concentration culture technology, higher concentration bacterial liquid is also suitable for the inactivation method of the invention.

In the inactivation method of the high-concentration bacterial ghost vaccine, when the viable count is reduced to 0CFU/ml, the culture is stopped.

Further, the inactivation method of the high-concentration ghost vaccine further comprises the steps of ghost preparation and culture, and the steps are as follows:

A) the bacterial ghost is prepared in a phage E protein-mediated cracking mechanism, a phage E gene is cloned into a PBV220 plasmid containing a temperature control element, a TC-E temperature control cracking element is amplified by taking PBV220-E as a template and is connected with a broad-spectrum plasmid pBBR1MCS-2, the plasmid is electrically transferred into competent cells of a brucella A19 strain, and the competent cells are screened, cultured and identified by a culture medium containing kanamycin to obtain a brucella A19-BG strain which meets the expectation;

B) carrying out propagation culture and seed preparation on the constructed A19-BG strain;

C) and (3) throwing the prepared seeds into a fermentation tank, and carrying out bacterial amplification culture at 28 ℃ at 300r/min for 48-56 h.

Further, the inactivation method of the high-concentration ghost vaccine further comprises a step of collecting inactivated antigens, wherein the steps are as follows: when the fermentation tank is inverted, all fermentation products in the aseptic fermentation tank are pumped into another aseptic fermentation tank, and the original antigen tank after the inversion is subjected to steam sterilization, so that the sterility of the tank body is ensured.

Further, the inactivation method of the high-concentration ghost vaccine further comprises a monitoring step of the inactivation condition of the antigen, and the monitoring step comprises the following steps: and sampling when the tank is inverted every time, and counting viable bacteria.

Compared with the prior art, the method has the following advantages: the bacterial liquid obtained by physical inactivation does not contain live bacteria, the surface of the treated bacterial strain has obvious cavities, is in a hollow shell shape, has complete shape and good immunocompetence, and an inactivator is not introduced, so that the subsequent storage of the bacterial strain is reducedAnd in the immunization step. Meanwhile, the inventor surprisingly discovers through experiments that the treatment method adopted by the invention can be used for culturing the bacterial liquid with the concentration of 4.5 multiplied by 10 ¹⁰ When the concentration is more than CFU/ml, the effect of completely removing live bacteria can be achieved, the method can be applied to large-scale industrial production of vaccines, the production cost is saved, and the biological safety risk is reduced.

In the present invention, the bacterium may be escherichia coli, brucella, salmonella, pasteurella, actinobacillus pleuropneumoniae, and in one embodiment of the present invention, the bacterium is brucella.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows bacterial liquid OD before and after inactivation in example 1 ₆₀₀ The result of the detection

FIG. 2 shows the results of the measurement of the growth curve of the A19BG strain

FIG. 3 shows the results of the detection of viable bacteria amount after treatment with different preparation methods in comparative example 1

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.

Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.

The steps of the construction of the plasmid and the design, amplification, enzyme digestion and ligation of the primers in the construction of the plasmid can be performed according to the molecular biology techniques known to those skilled in the art.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The main reagents and instrument information used in the embodiment of the invention are as follows:

TABLE 1 reagent and Instrument information Table

The technical effect of the technical scheme of the invention is illustrated by adopting the Brucella A19 as an example in the invention.

Example 1A 19BG Strain inactivation method

1. Propagation of basic seeds

Taking out the constructed strain in an ultra-low temperature refrigerator at minus 80 ℃, unfreezing the strain in a constant temperature incubator at 37 ℃, performing streak culture on a Brucella agar plate (kana plate 100 mu g/ml) by using a disposable inoculating loop after unfreezing, and culturing for 5-7 days at 28 ℃.

2 first order seed preparation

Single colonies were picked and placed in 10ml Brucella broth, incubated at 28 ℃ at 160r/min for 24 h.

3 second order seed preparation

The prepared primary seeds are taken and added into 200mL Brucella broth culture medium in a 500mL triangular shake flask according to the inoculation proportion of 2 percent, 100mg/mL kanamycin is taken and added into the culture medium according to the proportion of 1:1000, and the mixture is cultured for 24 hours at the temperature of 28 ℃ and at the speed of 300 r/min.

4 antigen culture

The bacterial liquid in the seeding tank is injected into a fermentation tank, 100mg/mL kanamycin is added into the Brucella broth according to the proportion of 1:1000, the mixture is cultured at 28 ℃ and 100r/min for more than 48 hours until the mixture reaches OD ₆₀₀ The value reaches 4.5 multiplied by 10 ¹⁰ CFU/ml is above; adding 5% sucrose solution when culturing for 18-24 h.

5 antigen inactivation culture

After the antigen culture is finished, raising the temperature of the tank body to 42 ℃, culturing at 300r/min, pumping all fermentation products in the sterile fermentation tank into another sterile fermentation tank at intervals of 24h, performing steam sterilization on the original antigen tank after the tank inversion is finished, ensuring the sterility of the tank body, and sampling and counting every day and OD (origin-to-population ratio) of the tank body ₆₀₀ And (5) monitoring the value.

6 detection of inactivation results

6.1 OD measurement results

Taking 5ml of inactivated antigen, adding the inactivated antigen into 5ml of Brucella broth, and detecting OD of bacterial liquid by using a spectrophotometer ₆₀₀ And recording the absorbance value of (a); culturing at constant temperature of 30 deg.C and 200r/min for 12 hr, and detecting OD again ₆₀₀ Value, OD ₆₀₀ The results of the detection are shown in FIG. 1.

The results show that OD was measured before and after culturing ₆₀₀ No change in value indicates complete inactivation of antigen.

6.2 coated sheet test results

Inactivating for 72h, taking 5ml of sample, inoculating 10ml of Brucella broth, culturing at 28 ℃ for 2 days, taking 200 μ l of the broth, culturing on a petri dish, and observing whether Brucella grows.

The results show that no Brucella grows on the cultured plate, indicating complete inactivation of the antigen.

EXAMPLE 2 selection of incubation duration and Reversal time intervals

The propagation and scale-up culture method of example 1 was used, and OD was performed every 8h ₆₀₀ Value detection, A19BG strain growth curve determination, the results are shown in figure 2.

The results show that the bacterial concentration has reached 4.7X 10 after 48h of culture ¹⁰ CFU/ml。

Then the concentration is 4.7 multiplied by 10 ¹⁰ The bacterial liquid of CFU/ml is subjected to inactivation treatment and viable bacteria counting, and specific test grouping and detection results are shown in Table 2.

TABLE 2 OD values at different reladling intervals

The results in table 2 show that, within a certain inactivation culture time, antigens can be completely inactivated at the reladling intervals of 8h, 12h and 24h, but the reladling time interval is short, which inevitably causes the reladling frequency to be increased, and the reladling interval time is properly prolonged on the basis of the minimum treatment time (8 h), so that the problem that the inactivation is not complete due to the difference of culture conditions in the viable bacteria removal effect of bacteria liquid with different concentrations in the production process can be avoided, and the production cost is increased due to too many reladling times, therefore, the inactivation culture time is determined to be 72h and the reladling interval is 24h by combining the viable bacteria removal effect, the immunogenicity and the production cost.

EXAMPLE 3 inactivation Effect of different bacterial liquid concentrations

The A19BG strain was antigen cultured using the propagation, amplification and inactivation culture methods of example 1-2, and OD was taken every 8h ₆₀₀ Value detection, the concentration of the bacterial liquid reaches 5.0 multiplied by 10 respectively ¹⁰ CFU/ml、5.5×10 ¹⁰ CFU/ml、6.0×10 ¹⁰ When CFU/ml is needed, the bacterial liquid is subjected to inactivation treatment and viable bacteria counting, and the inactivation effect of the invention is verified, so that the invention has the advantages ofThe results of the physical tests are shown in table 3.

TABLE 3 inactivation effect of different bacterial liquid concentrations

The results show that the inactivation method of the invention can be used for the bacterial liquid with the concentration of 5.0 multiplied by 10 ¹⁰ CFU/ml、5.5×10 ¹⁰ CFU/ml、6.0×10 ¹⁰ CFU/ml of the bacterial suspension was completely inactivated.

Comparative example 1

Preparation of A19BG bacterial liquid

The antigen culture of the A19BG strain was completed by the propagation and amplification culture method in example 1, and an A19BG bacterial solution was obtained.

2. Different method processing steps

2.1 method 1A 19BG bacterial liquid is inactivated at 42 ℃ and 300r/min for 72h, the tank switching operation is not carried out during the treatment, and sampling is carried out after 72h for detection.

2.2 method 2A 19BG bacterial liquid 42 deg.C, inducing lysis at 150r/min for 48h, adding beta-propiolactone (BLP) to the bacterial liquid, adding BLP again after 2h interval, making the final concentration 0.1% after 2 additions, continuing treatment at 150r/min for 36h at 42 deg.C, sampling and detecting.

2.3 inactivation was carried out according to the method of example 1 of the present invention.

3. Conclusion

The method for detecting viable bacteria in the bacterial liquid prepared by the method 1-2 is the same as that in the example 1, and the detection result is shown in figure 3; wherein the bacterial liquid treated by the method 1 still has 2.1 multiplied by 10 ² The existence of CFU/ml viable bacteria can not completely remove the viable bacteria, the viable bacteria of 16CFU/ml still exist in the bacterial liquid treated by the method 2, and the viable bacteria can not be completely removed, but the number of the viable bacteria in the bacterial liquid treated by the preparation method is 0, which shows that the preparation method has remarkable advantages in the aspect of preparing the bacterial ghost antigen with completely inactivated high-concentration antigen.

The ghost antigens prepared by the method in the comparative example 1 are used for preparing the A19BG vaccine with the same antigen content, and the protection effect of 3 vaccines after immunizing animals is detected, and the specific experimental grouping and the detection result are shown in the table 4.

Table 4 detection results of vaccine protection efficiency prepared by different treatment methods

According to the test results, the ghost prepared by the method can provide effective protective efficacy for the immune animals, the protective efficiency is up to 100%, the protective efficacy is up to 90% after the vaccine is prepared by the antigen without using the tank-inverting operation or introducing the inactivating agent for secondary inactivation treatment, and the protective efficacy of the invention is improved to 100%.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1.A method for inactivating a high-concentration ghost vaccine is characterized by comprising the following steps:

a) after the bacterial culture is finished, raising the temperature of the tank body, and performing inactivation culture;

b) replacing the fermentation tank at intervals of 8-72 h.

2. The inactivation method according to claim 1, wherein the antigen-inactivation culture time is 3 days in total.

3. The inactivation method according to claim 1, wherein the time interval between tank pourings in step b) is 12-36 h.

4. The inactivation method according to claim 3, wherein the period between tank pourings in step b) is 24 h.

5. The inactivation method according to claim 1, wherein the concentration of the bacterial liquid is not less than 4.5X 10 ¹⁰ CFU/ml。

6. The inactivation method according to claim 1, wherein the number of viable bacteria after inactivation is 0 CFU/ml.

7. The inactivation method according to claim 1, wherein the temperature of the tank body in the step a) is raised to 42 ℃ and the culture is carried out for 72 hours at 300 r/min; the operation step of replacing the fermentation tank once in the step b) is to collect all fermentation liquor in the fermentation tank and then pump the fermentation liquor into another sterile fermentation tank.

8. The inactivation method according to claim 1, further comprising a bacterial ghost preparation and culture step before step a).

9. The inactivation method of claim 8, wherein the step of preparing and culturing the bacterial ghost comprises:

A) the bacterial ghost is prepared in a phage E protein-mediated cracking mechanism, a phage E gene is cloned into a PBV220 plasmid containing a temperature control element, a TC-E temperature control cracking element is amplified by taking PBV220-E as a template and is connected with a broad-spectrum plasmid pBBR1MCS-2, the plasmid is electrically transferred into competent cells of a brucella A19 strain, and the competent cells are screened, cultured and identified by a culture medium containing kanamycin to obtain a brucella A19-BG strain which meets the expectation;

B) carrying out propagation culture and seed preparation on the constructed A19-BG strain;

C) and (3) throwing the prepared seeds into a fermentation tank, and carrying out bacterial amplification culture at 28 ℃ at 300r/min for 48-56 h.

10. Use of the inactivation method of any one of claims 1-9 in the preparation of a ghost vaccine.

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