AU2021100063A4 - A method for preserving Salmonella choleraesuis vaccine strain and special protective agents thereof - Google Patents

Abstract

The invention provides a method for preserving a Salmonella choleraesuis vaccine strain and a special protective agent. The present invention provides a method for preserving Salmonella choleraesuis vaccine strain under liquid nitrogen conditions by adding a protective agent to a Salmonella choleraesuis vaccine strain bacterial solution. The protective agent includes a permeable protective agent, semi-permeable protective agent, non-permeable protective agent and antioxidant, which is stored under liquid nitrogen conditions. The special protective agent has the effects of permeability, semi-permeability, non-permeability and anti-oxidation. There is no significant difference in the survival rate of the vaccine strain with different concentrations for different storage time under the selected protective agent formula, and the survival rate is maintained at 70% or more.

Description

A Method for Preserving Salmonella Choleraesuis Vaccine Strain and Special Protective Agents

thereof

Technical field

The invention belongs to the field of preparation technology of vaccine product auxiliary agents, and

specifically relates to a method for preserving C500/pGS/2SS vaccine strain and special protective agents thereof

Background art

Early-stage work of this invention is to prepare a vaccine strain C500/pGS/2SS (patent application number:

200810197981.8, strain deposit number: M208194), which is an attenuated Salmonella with double deletion of

asd and crp genes, including non-resistant screening hepatitis B surface antigen S gene and double copy

somatostatin (SS) gene plasmid, whose main function is to promote animal growth, is of great significance for

shortening the slaughter time of finishing pigs, improving feed utilization and reducing production costs.

Early stage studies of this invention have shown that the vaccine strain C500/pGS/2SS has achieved good

immune effects in mice, cattle, pigs and other animals, and at the same time has good safety (Liang Aixin 2009;

Shi Zhaoyi 2015; Zhang Jian 2010). The vaccine has now entered the stage of production testing for the safety of

agricultural genetically modified organisms. How to store the vaccine strain efficiently and stably for a long time

is an urgent problem to be solved.

Before filing this application, the applicant conducted a systematic study on the method of preserving

vaccine strain C500/pGS/2SS, and the results show that freeze-drying preservation method is a more suitable

preservation method (Li Chong, 2014), but the survival rate of the strain is low.In addition, the freeze-drying

preservation method also has disadvantages such as high equipment requirements, long production cycles, and

high product costs, and there is still a certain distance from commercial production. -130'C is the end point of

microbial mutation. Below this temperature, microorganisms are in a metabolically stagnant state and can be

stored for a long time. This principle is used for liquid nitrogen ultra-low temperature cryopreservation of strains.

Compared with the freeze-drying method, the damage during the liquid nitrogen cryopreservation method is

mainly freeze-thaw injury. In addition to the freeze-thaw injury during the freeze-drying method, there is also

drying damage. Therefore, use of the liquid nitrogen ultra-low temperature cryopreservation method often

achieves a better preservation effect. However, there are no reports on the research on liquid nitrogen ultra-low

temperature cryopreservation of Salmonella. In the process of liquid nitrogen ultra-low temperature

cryopreservation, it is necessary to maintain a suitable cooling rate. If the cooling rate is too fast, the intracellular

water will not have time to flow out of the cell, leading to a large number of intracellular ice crystals, which will increase the mechanical damage to the cell membrane and intracellular functional proteins. (Gwo et al 2005). The slow cooling rate will lead to excessive dehydration of cells, increase intracellular solute concentration, and increase solute damage (Fields et al 1997). In view of different cooling rates, there are three main methods commonly used for liquid nitrogen ultra-low temperature cryopreservation. They are one-step freezing method

(direct input of liquid nitrogen), two-step freezing method (-80°C-+liquid nitrogen), and three-step freezing

method (-20°C-*-80°C-*liquid nitrogen). Studies have shown that the two-step freezing method is a better

preservation method than one-step freezing method and three-step freezing method (Santiago-Vizquez et al

2007).

Protective agents can be divided into based on different types of effects: permeable protective agents,

semi-permeable protective agents, non-permeable protective agents and antioxidants. Permeable protective agents

can penetrate cell walls as well as cell membranes, and its main function is to prevent the formation of large ice

crystals in cells and solute damage caused by excessive dehydration of cells; while semi-permeable protective

agents can pass through cell walls but cannot pass through cell membranes, it is possible to partially dehydrate the

cells before freezing, thus the protective agent is concentrated between the cell wall and the cell membrane to

form a buffer layer, thereby mechanically protecting the cell; the non-permeable protective agent can neither

penetrate the cell wall nor the cell membrane, and its protective effect is mainly reflected outside the cells; the

main function of antioxidants is to prevent the oxidative deterioration of the strain during storage. Because

different types of protective agents have different protective effects, they are generally not used alone. Usually,

multiple types of protective agents can be combined to maximize the protection of the strains.

Summary of the invention

The task of the present invention is to overcome the shortcomings of the existing vaccine strain

C500/pGS/2SS preservation method, and develop a method suitable for long-term preservation of the vaccine

strain C500/pGS/2SS and a protective agent formula compatible with it, in order to achieve the objects of

long-term preservation of vaccine strain with high survival rate and large-scale production practice. The applicant

first used the survival rate of the vaccine strain C500/pGS/2SS as an indicator to study the protective effects of

several common permeable, semi-permeable, non-permeable protective agents and antioxidants at different

concentrations, and screened out the best protective agent and its concentration. Subsequently, the applicant set

three levels around the optimal concentrations of the four selected protective agents, and through orthogonal

design, the optimal protective agent formula was selected as: sucrose 2.5%, dextran 7%, dimethylacetamide 6%,

methionine 0.025%. Finally, under the condition of the best protective agent formula, the survival rate of different concentrations of vaccine strains under different storage time was studied, and it was found that after four weeks of storage, the survival rates of different concentrations of vaccine strains were all above 70%.

The objects of the present invention specifically include:

The first object of the present invention is to obtain a new method for preserving the vaccine strain

C500/pGS/2SS.

The second object of the present invention is to obtain a protective agent formula compatible with the new

preservation method.

The present invention is performed through the following technical solutions: (1) the protective agents of

different concentrations are mixed with the same volume of the 109 CFU/ml bacterial solution, put in a cryovial,

and put in liquid nitrogen after being placed at -80°C for 12 hours, and after storing for a week, the bacterial

solution is taken out and placed in a 37°C water bath for 30 minutes, and then the bacterial plate counting method

is used to detect the survival rate of the strains to screen out the best protective agent in each type of protective

agents and the concentration when the best preservation effect is obtained.

(2) Using the above freezing method, 3 concentration levels are set near the optimal concentration of the four

selected protective agents, the L 93 4 orthogonal test is designed, and the protection agent formula corresponding to

the freezing method is screened out through range analysis and variance analysis.

(3) Under the selected protective agent formula, the preservation rate of different concentrations of bacterial

solution after liquid nitrogen ultra-low temperature cryopreservation for one week and four weeks is tested for its

protective effect and storage stability.

The beneficial results of the present invention:

1. The invention obtains a new method for preserving vaccine strain C500/pGS/2SS. Compared with the

freeze-drying method, the liquid nitrogen ultra-low temperature cryopreservation method has lower equipment

requirements and easier operation, and more convenient promotion and application of the grassroots.

2. The present invention obtains a protective agent formula compatible with the liquid nitrogen ultra-low

temperature cryopreservation method. Compared with the freeze-drying preservation method, the liquid nitrogen

ultra-low temperature cryopreservation method is used to preserve the vaccine strain C500/pGS/2SS under the

protective agent formula, with higher strain survival rate and better storage stability.

Description of the drawings

Fig.1A shows the vaccine strain survival rate under different permeable protective agent at best concentration.

Fig.1B shows the vaccine strain survival rate under different semi-permeable protective agent at best concentration

Fig.1C shows the vaccine strain survival rate under different non-permeable protective agent at best concentration

Fig.2 shows electrophoresis of pVGS/2SS-asd plasmid digested by double restriction endonucleases after liquid

nitrogen preservation 4 weeks; wherein, Lane 1: DNA Maker; Lanes 2-3: the products of pVGS/2SS-asd plasmid

digested with EcoRI and HindIII.

Fig.3 shows the vaccine strain survival rate at different concentrations after four weeks

Specific modes for carrying out the embodiments

Example 1: screening of protective agents of liquid nitrogen ultra-low temperature cryopreserved vaccine

strain c500pGS/2SS

1.1 Fermentation and concentration of bacterial liquid

The bacterial solution identified by restriction digestion was inoculated in 5ml LB liquid medium according

to the ratio of 1:100, placed on a 37°C air bath constant temperature shaker for shaking culture at 200r/min for 14h,

and then the bacterial solution after shaking culture was inoculated in 350ml LB liquid medium according to the

ratio of 1:100, cultured with shaking at 37°C, 200r/min for 15h, centrifuged at 4000r/min for 10min to remove the

supernatant, and dissolved with sterile PBS buffer, the concentration of the bacterial solution was adjusted to

9CFU/ml.

1.2 Screening of protective agents of liquid nitrogen ultra-low temperature cryopreserved vaccine strain

c500pGS/2SS

The adjusted bacteria liquid was mixed with the following protective agents in an equal volume at a ratio of

1:1, and distributed in the cryovial, so that the concentration of each protective agent in the bacterial suspension

was finally shown in the following table:

Tablel-1 The concentration of protective agents

Type of protective Name of protective Concentration (%) agent agent 1 2

permeable protective ethylene glycol 3 6 9 12 15 agents NN-dimethylforma 3 6 9 12 15 mide N,N-dimethylacetam 3 6 9 12 15 ide semi-permeable trehalose 5 7.5 10 12.5 15 protective agents sucrose 5 7.5 10 12.5 15

lactose 5 7.5 10 12.5 15

mannitol 1 3 5 7 9

sodium alginate 2 1.5 1 0.5 0.25

non-permeable gelatin 1 2.5 5 7.5 10 protectiveagents polyvinylpyrrolidone 1 2.5 5 7.5 10

dextran 3 5 7 9 11

skimmed milk 5 10 15 20 25 bovine serum 5 7.5 10 12.5 15 albumin antioxidants Vc 0.05

L-sodium glutamate 0.05

methionine 0.05

1.3 Freezing and rewarming procedures

The freezing procedure was to place the bacterial suspension evenly mixed with the protective agent in a

cryovial at -80°C for 12 hours, and then put it in liquid nitrogen for one week. The process of resuscitation was to

immediately place the liquid nitrogen preserved bacterial solution in a 37 °C water bath for 30 minutes, and then

the survival rate of bacteria was detected through bacterial plate count.

1.4 Bacterial plate count

With the ultra-clean workbench, 0.5mL of the bacterial solution to be tested was taken and added to 4.5mL of

sterile PBS solution, a gradient dilution was performed in a ten-fold increasing ratio and three appropriate dilution

ratios were selected, and then lmL of the diluted bacterial solution was sucked up and added to the plate (the

diameter was 9cm), the LB solid medium dissolved and cooled to an appropriate temperature was added to the

plate and shaken evenly, and after solidification, it was incubated at 37°C for 48h at a constant temperature.

Finally, the number of colonies between 25 and 250 was selected for calculation, and the number of colonies was

multiplied by the dilution of the bacterial solution to calculate the average value to obtain the number of viable cells in the original bacterial solution.

1.5 Calculation of the survival rate of strains

Survival rate of strain(%)= viable count of bacteria after freezing /viable count of bacteria before freezing x

100%

1.6 Screening of permeable protective agent Tablel-2 The survival rate of vaccine strain in different permeable protective agents and concentration after one week (%) Protective Concentration (%) agent 15 12 9 6 3 ethylene 45.13±5.45A 44.66±0.40AB 31.46±5.92C 14.61±0.80D 35.11±0.40BC glycol dimethylforma 8.15±0.40A 28.09±3.18B 28.09±5.36B 26.03±1.41B 17.42±0.OC mide dimethylaceta 49.23±7.12A 40.42±7.95A 49.69±9.97A 50.29±13.43A 32.17±6.02A mide Notes: letters in the same row were totally different meant significant difference (P<0.05), and letters in the same row

were partially same meant no significant difference (P>0.05). The same was as follows.

It can be seen from Table 1-2 that ethylene ethylene glycol, dimethylformamide and dimethylacetamide had

the best preservation rates under the conditions of 15%, 9% and 6% respectively, which were 45.13%, 28.09%,

and 50.29% respectively. Combining Figure 1A, it can be found that under the optimal protective concentration,

the survival rate of vaccine strain between ethylene ethylene glycol, dimethylacetamide and dimethylformamide

was significantly different (P<0.05), while the survival rate of vaccine strain between ethylene glycol and

dimethylacetamide was not significantly different (P>0.05), but the preservation rate of dimethylacetamide

vaccine strain was higher.

1.7 Screening of semi-permeable protective agent Tablel-3 The survival rate of vaccine strain in different semi-permeable protective agent and concentration after one week (%) Protective Concentration No. agent 1 2 3 4 5 trehalose 46.07±4.90A 26.78±8.60B 40.26±1.17AB 33.89±9.99AB 31.65±11.93AB sucrose 34.24±1.12AC 29.40±8.19BC 41.57±0.79A 60.86±6.O1D 57.03±1.20D lactose - 39.35±5.56A 57.31±0.80B 47.48±1.99ABC 37.27±8.07C mannitol 54.78±1.20A 36.52±1.13B 42.70±1.59BC 17.60±5.76D 16.29±4.90D sodium 13.86±1.62A 9.55±2.45AB 12.91±5.88AB 8.80±0.65B 25.84±0.79C alginate Notes: The concentrations of trehalose, sucrose, lactose from 1 to 5 were 15%, 12.5%, 10%, 7.5%, 5%; the

concentrations of mannitol from 1 to 5 were 9%, 7%, 5%, 3%, 1%; the concentrations of sodium alginate from 1

to 5 were 2%,1.5%,1%,0.5%,0.25%.

From the analysis in Table 1-3, it can be seen that trehalose and mannitol achieved better preservation effects in the high concentration range, while sucrose and lactose achieved better preservation effects in the lower concentration range. Trehalose, sucrose, lactose, mannitol and sodium alginate achieved best preservation effects at the concentrations of 15%, 7.5%, 10%, 9% and 0.25% respectively. The best preservation rates were 46.07%,

60.86%, 57.31%, 54.78% and 25.84%, respectively. Combining Table 1-3 and Figure IB, it can be found that

7.5% sucrose had the best preservation effect, followed by lactose and mannitol, but the difference between the

three was not significant (P>0.05), and the best preservation rate of sucrose was significantly higher than that of

trehalose and sodium alginate (P<0.05), there was no significant difference in the preservation rate of trehalose,

lactose and mannitol (P>0.05), and the preservation effect of sodium alginate was significantly lower than that of

other protective agents ( P<0.05).

1.8 Screening of non-permeable protective agents

Tablel-4 The survival rate of vaccine strain under different non-permeable protective agents and concentration after one week (%) Protective agent Concentration No. 1 2 3 4 5 gelatin 38.77±1.59A 19.29±4.54B 22.47±0.98BC 26.97±9.88BC 20.23±1.59BC PVP 13.48±6.08A 22.85±7.33B 19.66±3.68AB 19.29±3.1OAB 19.29±5.34AB dextran 41.95±5.65A 19.10±0.56B 40.17±1.99AC 42.14±2.38AC 45.23±12.31AC skimmed milk 19.66±0.79A 44.95±2.38B 40.17±l.18B 43.54±0.40B 20.97±4.58AC powder bovine serum 23.88±0.40A 15.73±1.95B 30.06±0.40C 19.29±2.66B 19.39±1.99BC albumin Notes: the concentration No. was from 1 to 5, the concentration of gelatin, PVP were 10%, 7.5%, 5%, 2.5%, 1%,

the concentration of dextran was 11%, 9%, 7%, 5%,2.5%, the concentration of skimmed milk powder was 25%,

20%,15%,10%,5%, the concentration of bovine serum albumin was 5%,3.75%,2.5%,1.25%,0.5%.

From the analysis in Table 1-4, it can be seen that gelatin, PVP, dextran, skimmed milk powder and bovine

serum albumin achieved the best preservation rates at the concentrations of 10%, 7.5%, 2.5%, 20%, and 2.5%,

respectively. The preservation rates were 38.77%, 22.85%, 45.23%, 44.95% and 30.06%. Combining Figure 1-3,

it can be found that the non-permeable protective agents with better preservation effects at the optimal

concentration were dextran, skimmed milk powder and gelatin, and the difference between the three was not

significant (P>0.05), but the preservation rate of dextran and skimmed milk powder was higher than that of gelatin.

The preservation rate of PVP and bovine serum albumin was significantly lower than that of dextran and skimmed

milk powder (P<0.05). Therefore, among the non-permeable protective agents, 2.5% dextran and 20% skimmed

milk powder were better protective agents.

1.9 Screening of antioxidants

Table 5 The survival rate of vaccine strain under different antioxidants after one week(%)

Protective agent Concentration (%) 0.05 Vc 3.56±0.86A methionine 5.24±1.72A L-sodium 3.18±1.17A glutamate

In order to improve the stability of the vaccine strain C500 (pVGS/2SS-asd) during storage, it was necessary

to add antioxidants to the protective agent formula. Table 1-5 showed that the preservation rate of methionine was

the highest at 5.24%, but compared with other antioxidants, there was no significant difference (P>0.05).

Example 2: Compatibility optimization of protective agent of liquid nitrogen low temperature cryopreserved

vaccine strain C500/pGS/2SS

2.1 Orthogonal design experiment arrangement

Table2-1 L9(3 4) Orthogonal design table

Levels Factors sucrose dextran dimethylacetami methionine de 1 7.5% 9% 9% 0.075% 2 5% 7% 6% 0.05% 3 2.5% 5% 3% 0.025%

Table2-2 The arrangements of liquid nitrogen preservation multi-factor process optimization experiments

Line 1 Line 2 Line 3 Line 4 Experiment Sucrose (%) Dextran (%) Dimethylacetamide Methionine NO. (%) (%) 1 2.5(A3) 7(B2) 3(C3) 0.075(D1) 2 2.5 5(B3) 9(C1) 0.05(D2) 3 5(A2) 9(B1) 3 0.05 4 5 5 6(C2) 0.075 5 5 7 9 0.025(D3) 6 7.5(A1) 5 3 0.025 7 7.5 9 9 0.075 8 2.5 9 6 0.025 9 7.5 7 6 0.05 The adjusted bacterial solution was mixed with the above protective agent formula in an equal volume at a

ratio of 1:1 to make the final concentration as shown in the table above, which was divided into cryovials,

operations were repeated three times for each group, after being placed at -80°C for 12h and being put into liquid

nitrogen for a week, then the bacterial solution stored in liquid nitrogen was immediately put in a 37°C water bath for 30 minutes, and then the survival rate of the bacteria was detected to screen out the protective agent formula with the best protection effect. 2.2 Screening of protective agent formula of liquid nitrogen low temperature cryopreserved vaccine strain C500/pGS/2SS

Table2-3. The range analysis table of liquid nitrogen preservation multi-factor process optimization experiments Experi Factors Preservation ment A(Sucrose) B(Dextran) C(Dimethylacetami D(Methionine) rate (%) NO. de) 1 3 2 3 1 70.54±3.55 2 3 3 1 2 40.75±2.62 3 2 1 3 2 48.24±5.16 4 2 3 2 1 45.18±5.99 5 2 2 1 3 55.43±4.93 6 1 3 3 3 42.33±2.99 7 1 1 1 1 32.56±4.10 8 3 1 2 3 72.81±1.99 9 1 2 2 2 70.93±4.94 KI 48.61 51.20 42.91 49.43 K2 49.65 65.63 62.97 53.31 K3 61.37 42.75 53.70 56.86 range 12.76 22.88 20.06 7.43 optimal A3 B2 C2 D3 levels sorting dextran>dimethylacetamide>sucrose>methionine

Table 2-4. The analysis of variance table of liquid nitrogen preservation multi-factor process optimization experiments

Source Sumof type III DF Mean square F value Sig. squares

correction 5082.733a 10 508.273 30.715 0.000 model intercept 60163.586 1 60163.586 3635.746 0.000 sucrose 843.390 2 421.695 25.483 0.000 dextran 2054.488 2 1027.244 62.077 0.000 dimethylaceta 1511.803 2 755.901 45.680 0.000 mide methionine 270.281 4 67.570 4.083 0.021

error 231.669 14 16.548

total 73952.114 25

total correction 5314.402 24

a. R square = 0.956 (adjusted R square = 0.925)

From the range analysis table in Table 2-3, it can be seen that the combination A3B2C2D3 group had the best

protective effect. Combined with the experimental variance analysis in Table 2-4, it can be seen that the levels of

sucrose, dextran, and dimethylacetamide were extremely significant (P<0.01). The levels of methionine were

significantly different (P<0.05), indicating that these 4 factor levels were the optimal levels and cannot be 1I replaced by other levels. After comprehensive considerations, the optimal protective agent combination for liquid nitrogen low temperature cryopreservation of the vaccine strain C500 (pVGS/2SS-asd)is A3B2C2D3, that is, the vaccine strain C500 (pVGS/2SS-asd) under the conditions of 2.5% sucrose, 7% dextran, 6% dimethylacetamide, and 0.025% methionine had the best preservation effect.

Example 3: Detection of storage stability of vaccine strain C500/pGS/2SS

3.1 Experimental design

Under the best protective agent formula that was screened out, different concentrations of vaccine strain

C500/pGS/2SS were tested, and their storage stability for 1 week and 4 weeks was tested, and the plasmid

stability was tested at the end of the experiment.

3.2 Plasmid extraction and restriction digestion identification

On the ultra-clean workbench, a pipette was used to take 1ml of bacterial solution, and the plasmids were

extracted according to the instructions of the Beijing Tiangen Plasmid Small Extraction Kit. The specific steps

were as follows:

(1) 1.5 mL of bacterial solution was placed in a 2 mL clean centrifuge tube, centrifuged at 12000 rpm for 1

min, the supernatant was discarded, and the bacterial sediment was collected.

(2) 250pLP1 was added to the centrifuge tube with bacterial precipitate, the bacterial precipitate was

resuspended, and vortexed until the bacteria was completely suspended.

(3) Another 250gLP2 was added to lyse the bacteria and the bacteria was completely lysed after gently

turning upsidedown 10 times.

(4) Then 350pL of solution P3 was added to the centrifuge tube, after immediately turning upside down

gently 6-8 times, a white flocculent precipitate will appear, which was centrifuged at 12000rpm for 10min.

(5) The supernatant collected in the previous step was transferred to a centrifugal adsorption column, and

centrifuged at 12000 rpm for 1 min, and the collection tube waste was discarded.

(6) 600 pL of rinsing solution was added to the centrifugal adsorption column, centrifuged at 12000 rpm for

1 min, and the waste collection tube was discarded; the rinsing step was repeated once, the collection tube waste

was discarded, and after centrifuging at 2000 rpm for 2 min, the rinsing fluid WB was removed as much as

possible.

(7) The adsorption column was placed into a clean centrifuge tube, 30 pLEB eluent was added to the center

of the adsorption column, maintained at room temperature for 2 minutes, and centrifuged at 12000 rpm for 1

minute.

(8) The elated liquid was re-added to the adsorption column and the above steps were repeated. the plasmid

solution was collected into a centrifuge tube.

(9) Restriction enzyme digestion identification of plasmid

The extracted plasmids were identified by restriction enzyme digestion, and the restriction enzyme digestion

system was as follows: Reagents Volume (uL) ddH20 (sterilized) 6 lOxM Buffer 2 Hind III 1 EcoR I 1 plasmids 10 total volume 20

In a 37°C constant temperature water bath for 2 hours, 1% agarose gel electrophoresis was used for detection,

and the result of restriction enzyme digestion was observed on a gel imaging system instrument.

3.3 Preservation stability of vaccine strain C500/pGS/2SS at different concentrations

Table3-1. The survival rate of vaccine strain at different concentrations in different periods

concentration(cfu/mL) 1W 4W

109 77.33±9.30A 73.80±9.34A

10i 74.20±6.25A 73.61±7.97A

107 70.80±9.90A 71.80±9.20A

It can be seen from Table 3-1 that after storing the vaccine strain according to the optimal protective agent

formula selected, the vaccine strain achieved a higher preservation rate at each concentration, and with the

extension of the storage time, the survival rate of the vaccine strain had no obvious change (P>0.05), showing

good storage stability. Combining with Figure 3, it can be seen that the survival rate of different concentrations of

vaccine strain after liquid nitrogen low temperature cryopreservation was not significant (P>0.05). The above

results indicated that liquid nitrogen low temperature cryopreservation can be applied to the preservation of

vaccine strain C500 (pVGS/2SS-asd), especially had great advantages in the long-term preservation of vaccine

strain C500 (pVGS/2SS-asd), and in suitable concentration range, the survival rate of vaccine strain did not

change significantly.

3.4 Plasmid stability detection of vaccine strain C500/pGS/2SS after liquid nitrogen low temperature

cryopreservation

As can be seen from Figure 2, the plasmids extracted after liquid nitrogen low temperature cryopreservation of

the vaccine strain C500 (pVGS/2SS-asd was digested with EcoRI and HindIII, and a band of about 780 bp appeared,

which was the same as the target band (S/2SS) was consistent, indicating that the plasmids of the vaccine strain can

still maintain good stability after liquid nitrogen ultra-low temperature cryopreservation, and there was no genetic

mutation.

Claims (2)

What is claimed is:

1. A method for preserving Salmonella choleraesuis vaccine strain, wherein, a protective agent is added to a

bacterial solution of Salmonella choleraesuis vaccine strain in a volume ratio of 1:1, and the components of the

protective agent are sucrose ratio 2.5 wt%, dextran 7 wt%, dimethylacetamide 6 wt% and methionine 0.025 wt%,

which is stored under liquid nitrogen conditions.

2. A special protective agent for preserving Salmonella choleraesuis vaccine strain under liquid nitrogen

conditions, wherein the components of the protective agent are sucrose 2.5 wt%, dextran 7 wt%, and

dimethylacetamide 6 wt% and methionine 0.025 wt%.

preservation rate(%) preservation rate(%)

1 of 3 1 Figure 1B Figure 1A Drawings

preservation rate(%)

2 of 3 2 Figure 2 Figure 1C

preservation rate(%)

3 of 3 3 Figure 3 concentration