Freeze-drying preparation method of haemophilus influenzae type b

Abstract

The invention discloses a freeze-drying preparation method of haemophilus influenzae type b, which comprises the following steps: S1, dissolving a haemophilus influenzae type b cryopreserved strain with normal saline, and recovering the haemophilus influenzae type b cryopreserved strain; s2, inoculating the resuscitation bacteria obtained in the step S1 into a new chocolate blood agar culture medium, and culturing in a CO2 incubator for 10-20 hours; s3, flushing the lawn on the surface of the proliferation bacterium culture medium obtained in the step S2 by using a skim milk solution, moving out a bacterium solution obtained by flushing, uniformly shaking, and sub-packaging into strain tubes; and S4, adding a cryopreservation solution with a specific proportion into the strain tube in which the strains are subpackaged, which is obtained in the step S3, uniformly shaking, sealing the strain tube with absorbent cotton, performing programmed cooling at the temperature of-80 DEG C, then sealing the orifice of the strain tube to obtain the freeze-dried b-type haemophilus influenzae strain, and performing cryopreservation on the freeze-dried b-type haemophilus influenzae strain. The method is suitable for long-term preservation of strains, the preservation activity is well maintained, and the fermentation culture yield after recovery is high.

Description

Technical Field

[0001] The present application relates to the technical field of bacterial strain preparation, and in particular to a freeze-drying preparation method of Haemophilus influenzae type b. Background Art

[0002] Haemophilus influenzae Hib is an important pathogen that causes primary respiratory infections in infants and young children and secondary infections after viral infections. According to the composition and antigenicity of the capsular polysaccharide, it can be divided into six serotypes: a, b, c, de, and f. Haemophilus influenzae type b (Haemop hilus influenzae type b Hib) is the type that is most harmful to humans. Hib is a type of Gram-negative bacillus. It is a pathogen that can cause local and invasive infections such as purulent meningitis, pneumonia, epiglottitis, and purulent arthritis in infants and young children. It is more common in infants and young children under 5 years old. In cases of purulent meningitis in children, the mortality rate can reach 3% to 5%, and 15% to 30% of the survivors have varying degrees of neurological sequelae.

[0003] Hib is very harmful and its treatment is relatively complicated, so vaccination is the best way to prevent the vast majority of Hib cases. Hib vaccine is produced by covalently binding purified Hib capsular polysaccharide to binding protein, and Hib capsular polysaccharide is extracted from fermented Haemophilus influenzae type b. Therefore, in the production of Hib vaccine, the strain of Haemophilus influenzae type b is the core preparation raw material, and the preparation and preservation of Haemophilus influenzae type b is an important part of Hib vaccine production.

[0004] In the existing Hib vaccine production process, most strains of Haemophilus influenzae type b are obtained from the China Medical Bacteria Collection Administration Center. Domestic vaccine manufacturers currently do not have the relevant technology to prepare Haemophilus influenzae type b strains. The Haemophilus influenzae type b strains obtained from the China Medical Bacteria Collection Administration Center are used in Hib vaccine production, but the fermentation yield is low. Summary of the invention

[0005] In order to solve at least one of the above technical problems, a method for preparing a strain of Haemophilus influenzae type b that is suitable for long-term storage of the strain, maintains good storage activity, and has a high fermentation culture yield after recovery is developed. The present application provides a freeze-drying preparation method for Haemophilus influenzae type b.

[0006] The present application provides a freeze-drying preparation method of Haemophilus influenzae type b, comprising the following steps:

[0007] S1. Dissolve the frozen strain of Haemophilus influenzae type b with physiological saline, then place the dissolved bacterial solution in chocolate blood agar medium, and culture it in a CO2 incubator at 35-37°C for 16-24h to obtain a resuscitation strain culture medium;

[0008] S2, rinsing the bacterial lawn on the surface of the resuscitation strain culture medium obtained in step S1 with physiological saline, inoculating the rinsed bacterial solution into a new chocolate blood agar culture medium, maintaining the temperature at 35-37° C., and culturing in a CO2 incubator for 10-20 hours to obtain a proliferation culture medium;

[0009] S3, washing the bacterial lawn on the surface of the culture medium for proliferating bacteria obtained in step S2 with a skim milk solution, removing the bacterial solution obtained by washing, shaking it well, and then dispensing it into bacterial tubes to obtain bacterial tubes for dispensing bacterial strains;

[0010] S4, adding freezing solution to the culture tube of the subpackaged culture obtained in step S3, shaking well, sealing the culture tube with absorbent cotton, cooling by program at -80°C, and then sealing the tube mouth of the culture tube to obtain freeze-dried Haemophilus influenzae type b strains, and freezing and storing;

[0011] In step S4, the mass fraction ratio of each component of the freezing solution includes: DMEM / F12 culture medium 40-50%, trehalose 6-10%, dimethyl sulfoxide 12-16%, glucose 6-8%, lecithin 2-4%, and the balance is fetal bovine serum.

[0012] By adopting the above technical scheme, the present application adopts a frozen strain of Haemophilus influenzae type b, and resuscitates it through a specific resuscitation step and a specific chocolate blood agar medium, which can effectively ensure that the remaining live Haemophilus influenzae type b bacteria after resuscitation have good activity and are suitable for subsequent proliferation and culture; the present application adopts a specific chocolate blood agar medium and a specific culture step to proliferate and culture the revived Haemophilus influenzae type b, which can further improve the live bacteria concentration and live bacteria activity in the obtained bacterial liquid; the present application adopts a specific packaging method and a specific freezing solution to freeze-dry the Haemophilus influenzae type b obtained by the proliferation culture, and then seals and freezes it, which can effectively ensure that the frozen Haemophilus influenzae type b remains active and effectively ensure the quality of the prepared Haemophilus influenzae type b strain.

[0013] Optionally, in step S1 and step S2, the concentration ratio of each component of the chocolate blood agar culture medium includes: 20-25 g / L bovine casein peptone, 15-20 g / L meat peptone, 40-60 g / L corn starch, 3-5 g / L sodium chloride, 14-18 g / L agar, 400-500 mg / L multivitamins, and 80-100 g / L defibrinated bovine blood.

[0014] Optionally, in step S1, dissolving with physiological saline is performed by adding physiological saline and then repeatedly blowing and dissolving.

[0015] Optionally, in step S2, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the incubator is 4.5-5.5%.

[0016] Optionally, in step S2, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the incubator is 4.5-5.5%.

[0017] Optionally, in step S3, during packaging, the packaging volume per tube is controlled to be 0.2-0.4 mL.

[0018] Optionally, in step S4, the opening of the culture tube is sealed by hot-melt sealing.

[0019] Optionally, in step S4, the cryopreservation is performed by cryopreservation in liquid nitrogen.

[0020] Optionally, after step S1, the bacterial moss in the culture medium of the revived strain needs to be sampled and identified before proceeding to step S2.

[0021] Optionally, in step S2, after that, it is necessary to sample and identify the bacterial moss in the culture medium of the revived strain before proceeding to step S3.

[0022] In summary, the present invention includes at least one of the following beneficial technical effects:

[0023] 1. The present application uses a frozen strain of Haemophilus influenzae type b, which is revived through specific resuscitation steps and specific chocolate blood agar medium, which can effectively ensure that the remaining live Haemophilus influenzae type b bacteria after resuscitation have good activity and are suitable for subsequent proliferation and culture.

[0024] 2. The present application adopts a specific chocolate blood agar medium and specific culture steps to carry out proliferation culture of the revived Haemophilus influenzae type b, which can further increase the concentration and activity of live bacteria in the obtained bacterial solution.

[0025] 3. The present application adopts a specific packaging method and a specific freezing solution to freeze-dry the Haemophilus influenzae type b obtained by proliferation culture, and then seal and freeze it, which can effectively ensure that the frozen Haemophilus influenzae type b remains active and effectively ensure the quality of the prepared Haemophilus influenzae type b strain. DETAILED DESCRIPTION

[0026] The present application is further described in detail below with reference to the embodiments.

[0027] The present application designs a freeze-drying preparation method of Haemophilus influenzae type b, comprising the following steps:

[0028] S1. Dissolve the frozen strain of Haemophilus influenzae type b with physiological saline, then place the dissolved bacterial solution in chocolate blood agar medium, and culture it in a CO2 incubator at 35-37°C for 16-24h to obtain a resuscitation strain culture medium;

[0029] S2, rinsing the bacterial lawn on the surface of the resuscitation strain culture medium obtained in step S1 with physiological saline, inoculating the rinsed bacterial solution into a new chocolate blood agar culture medium, maintaining the temperature at 35-37° C., and culturing in a CO2 incubator for 10-20 hours to obtain a proliferation culture medium;

[0030] S3, washing the bacterial lawn on the surface of the culture medium for proliferating bacteria obtained in step S2 with a skim milk solution, removing the bacterial solution obtained by washing, shaking it well, and then dispensing it into bacterial tubes to obtain bacterial tubes for dispensing bacterial strains;

[0031] S4, adding freezing solution to the culture tube of the subpackaged culture obtained in step S3, shaking well, sealing the culture tube with absorbent cotton, cooling by program at -80°C, and then sealing the tube mouth of the culture tube to obtain freeze-dried Haemophilus influenzae type b strains, and freezing and storing;

[0032] In step S4, the mass fraction ratio of each component of the freezing solution includes: DMEM / F12 culture medium 40-50%, trehalose 6-10%, dimethyl sulfoxide 12-16%, glucose 6-8%, lecithin 2-4%, and the balance is fetal bovine serum.

[0033] Specifically, in step S1 described in the present application, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the box is 4.5-5.5%; in step S2 described in the present application, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the box is 4.5-5.5%.

[0034] The main differences between the present application and the prior art are as follows. First, the present application does not use the type b Haemophilus influenzae strain directly obtained from the market or from the China Medical Bacteria Collection Management Center as the raw material for the preparation of Hib vaccine, but uses specific steps to cultivate the type b Haemophilus influenzae bacterial solution with high activity and concentration, and then uses specific freezing solution and freeze-drying steps to prepare high-quality type b Haemophilus influenzae strains. In this way, high-quality strain raw materials can be effectively obtained, thereby effectively improving the fermentation yield of type b Haemophilus influenzae in the subsequent preparation of Hib vaccine, and effectively improving the vaccine yield.

[0035] The following are preparation examples and embodiments of the present application.

[0036] In the preparation examples and embodiments of the present application, the sources of the main raw materials are as follows:

[0037] Haemophilus influenzae type b strain was obtained from the Chinese Medical Bacteria Collection Center; bovine casein peptone and meat peptone were purchased from Shanghai Xige Biotechnology Co., Ltd.; glucose and agar were purchased from Shanghai Yaji Biotechnology Co., Ltd.; multivitamins, using PolyViteX 55652 from Mérieux, France, were purchased from Quzhou Xinzhi Biotechnology Co., Ltd.; defibrinated bovine blood was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DMEM / F12 medium was purchased from Thermo Fisher Scientific; lecithin was purchased from Beijing Ita Biotechnology Co., Ltd.; fetal bovine serum was purchased from Thermo Fisher Scientific; and chocolate blood agar medium was purchased from Shanghai Guandao Bioengineering Co., Ltd.

[0038] Preparation Examples 1 to 6

[0039] Preparation Example 1

[0040] The components of the freezing solution of this preparation example include: 40% DMEM / F12 culture medium, 6% trehalose, 12% dimethyl sulfoxide, 6% glucose, 2% lecithin, and the balance is fetal bovine serum.

[0041] When preparing, mix the components according to the above ratio and shake well.

[0042] Preparation Example 2

[0043] The components of the freezing solution of this preparation example include: 50% DMEM / F12 culture medium, 10% trehalose, 16% dimethyl sulfoxide, 8% glucose, 4% lecithin, and the balance is fetal bovine serum.

[0044] When preparing, mix the components according to the above ratio and shake well.

[0045] Preparation Example 3

[0046] The components of the freezing solution of this preparation example include: 48% DMEM / F12 culture medium, 8% trehalose, 14% dimethyl sulfoxide, 7.5% glucose, 2.8% lecithin, and the balance is fetal bovine serum.

[0047] When preparing, mix the components according to the above ratio and shake well.

[0048] Preparation Example 4

[0049] The concentration ratio of each component of the chocolate blood agar medium of this preparation example includes: 20 g / L bovine casein peptone, 15 g / L meat peptone, 40 g / L corn starch, 3 g / L sodium chloride, 14 g / L agar, 400 mg / L multivitamins, and 80 g / L defibrinated bovine blood.

[0050] During configuration, all components can be evenly mixed according to the above ratio.

[0051] Preparation Example 5

[0052] The concentration ratio of each component of the chocolate blood agar medium of this preparation example includes: 25 g / L bovine casein peptone, 20 g / L meat peptone, 60 g / L corn starch, 5 g / L sodium chloride, 18 g / L agar, 500 mg / L multivitamins, and 100 g / L defibrinated bovine blood.

[0053] During configuration, all components can be evenly mixed according to the above ratio.

[0054] Preparation Example 6

[0055] The concentration ratio of each component of the chocolate blood agar medium of this preparation example includes: 22 g / L bovine casein peptone, 18 g / L meat peptone, 54 g / L corn starch, 4 g / L sodium chloride, 16 g / L agar, 450 mg / L multivitamins, and 95 g / L defibrinated bovine blood.

[0056] During configuration, all components can be evenly mixed according to the above ratio.

[0057] Examples 1 to 8

[0058] Example 1

[0059] The freeze-drying preparation method of Haemophilus influenzae type b of this embodiment comprises the following steps:

[0060] S1. Add 0.8 mL of physiological saline to the culture tube of the frozen strain of Haemophilus influenzae type b, repeatedly blow and dissolve it, and then place the dissolved bacterial solution in a commercially available chocolate blood agar medium, and culture it at 37°C in a CO2 incubator with a CO2 concentration of 3% for 16 hours to obtain a resuscitated strain culture medium; take a bacterial sample and examine it under a Gram staining microscope: the appearance of the strain should be Gram-negative short rods (red), and after observing at least 10 fields of view without miscellaneous bacteria or atypical bacteria, the identification meets the requirements;

[0061] S2, rinse the bacterial lawn on the surface of the culture medium of the revived strain obtained in step S1 with physiological saline, inoculate the bacterial solution obtained by rinsing into a new commercially available chocolate blood agar culture medium, maintain the temperature at 37°C, and culture for 10 hours in a CO2 incubator under the condition of 3% CO2 concentration to obtain a culture medium for proliferating bacteria; take a bacterial sample and examine it under Gram staining microscopy: the appearance of the bacterial species should be Gram-negative short rods (red), and after observing at least 10 fields of view without miscellaneous bacteria or atypical bacteria, it is identified as meeting the requirements;

[0062] S3, washing the bacterial moss on the surface of the culture medium obtained in step S2 with a 10% skim milk solution, transferring the washed bacterial solution to a flask, shaking it well, and dispensing it into bacterial tubes, with each tube encapsulating 0.2 mL, to obtain bacterial tubes for dispensing bacterial strains;

[0063] S4. Add 1.0 mL of the freezing solution of Preparation Example 1 to the culture tube of the subpackaged culture obtained in step S3, shake well, seal the culture tube with absorbent cotton, cool to -80°C, and then seal the tube mouth of the culture tube with hot melt to obtain the freeze-dried Haemophilus influenzae type b strain, which is placed in liquid nitrogen for cryopreservation.

[0064] Example 2

[0065] The difference between this embodiment and embodiment 1 is that in step S4, the freezing solution of preparation example 2 is used.

[0066] Example 3

[0067] The difference between this embodiment and embodiment 1 is that in step S4, the freezing solution of preparation example 3 is used.

[0068] Example 4

[0069] The difference between this embodiment and embodiment 3 is that in step S1 and step S2, the chocolate blood agar medium of preparation example 4 is used.

[0070] Example 5

[0071] The difference between this embodiment and embodiment 3 is that in step S1 and step S2, the chocolate blood agar medium of preparation example 5 is used.

[0072] Example 6

[0073] The difference between this embodiment and embodiment 3 is that in step S1 and step S2, the chocolate blood agar medium of preparation example 6 is used.

[0074] Example 7

[0075] The difference between this embodiment and embodiment 6 is that in step S1, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the incubator is 5%; in step S2, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the incubator is 5%.

[0076] Example 8

[0077] The difference between this embodiment and embodiment 7 is that in step S3, 0.4 mL is packaged in each tube; and in step S4, 1.2 mL of freezing solution is added to each tube.

[0078] Comparative Example

[0079] This comparative example uses a frozen strain of Haemophilus influenzae type b from the China Medical Bacteria Collection Center.

[0080] Detection of bacterial growth rate

[0081] The frozen strains of Haemophilus influenzae type b were prepared by the preparation methods of Examples 1 to 8 of the present application. The frozen strains of Haemophilus influenzae type b prepared in Examples 1 to 8 of the present application and the frozen strains of Haemophilus influenzae type b of the comparative example were taken, 1 mL of physiological saline was added, and the mixture was repeatedly pipetted and dissolved, and the OD of the dissolved solution was detected. 600 and bacterial turbidity.

[0082] The frozen strains of Haemophilus influenzae type b prepared in Examples 1 to 8 of the present application and the frozen strains of Haemophilus influenzae type b of the comparative example were soaked and revived in a resuscitation solution of German Nobimpex brand, product number S201-100, and then fermented to produce Haemophilus influenzae type b bacterial solution.

[0083] The concentration ratio of each component of the fermentation liquid used is: bovine casein peptone 22g / L, meat peptone 18g / L, glucose 75g / L, dipotassium hydrogen phosphate 8g / L, sodium chloride 4g / L, hemoglobin 120mg / L, agar 900mg / L, vitamin C 450mg / L, and penicillin 40mg / L; the solvent of the culture liquid is pure water.

[0084] Fermentation steps: inoculate the revived Haemophilus influenzae type b into a 20 L fermentation tank with fermentation liquid added, with an inoculation rate of 5%, adjust the pH to 7.5, and culture at 37°C and 500 rpm with stirring for 12 hours.

[0085] Detect the OD of the fermentation broth 600 and bacterial turbidity.

[0086] The results are shown in Table 1 below.

[0087] Table 1 Test results of Examples 1 to 8 and Comparative Examples

[0088] <![CDATA[OD 600 ]]> Bacterial turbidity (MCF) <![CDATA[OD after fermentation 600 > Bacterial turbidity after fermentation (MCF) Example 1 5.02 10.74 6.59 15.89 Example 2 5.08 10.79 6.61 15.95 Example 3 5.11 10.84 6.68 16.17 Example 4 5.22 11.07 6.82 17.19 Example 5 5.24 11.11 6.85 17.28 Example 6 5.27 11.19 6.91 17.42 Example 7 5.36 11.31 6.98 17.86 Example 8 5.41 11.62 7.02 18.11 Comparative Example 5.42 11.76 5.72 13.48

[0089] It can be seen from the data in Table 1 that the Haemophilus influenzae type b strains prepared by the methods of Examples 1 to 8 of the present application are compared with the frozen strains of Haemophilus influenzae type b in the existing China Medical Bacteria Collection Center. On the premise that the total bacterial concentration after resuscitation is roughly the same, the fermentation growth rate of the Haemophilus influenzae type b strains prepared by the methods of Examples 1 to 8 of the present application is significantly improved compared with the frozen strains of Haemophilus influenzae type b in the China Medical Bacteria Collection Center, and the yield is significantly improved.

[0090] From the data comparison of Examples 1 to 8 in Table 1, it can be seen that after the ratio of the freezing solution of the present application is optimized, the fermentation value-added rate can be further improved. In addition, the present application adopts a specific chocolate blood agar medium and optimizes the culture conditions to obtain the Haemophilus influenzae type b strain, and the fermentation value-added rate can also be further improved.

[0091] The applicant conducted multiple groups of experiments in step S1 and step S2 of the present application, with the culture conditions within the scope of the claims of the present application; the culture conditions were within the scope designed in the present application, and the fermentation growth rates of the obtained Haemophilus influenzae type b strains were not much different.

[0092] Bacteria recovery activity test

[0093] Total bacterial count determination:

[0094] Take the strain tubes of the frozen strains of Haemophilus influenzae type b prepared in Examples 1 to 8 of the present application and the frozen strains of Haemophilus influenzae type b of the comparative example, add 1 mL of physiological saline, and repeatedly blow and beat to dissolve to obtain a bacterial solution; 0.1 mL of the bacterial solution from each strain tube is taken, diluted 10 times with physiological saline, and single cell counting and analysis are performed using a flow cytometer, and then the total number of bacteria in each strain tube is calculated based on the dilution multiple and the sampling volume ratio.

[0095] Determination of viable count:

[0096] Take 0.1 mL of sample from each of the dissolved and diluted strain tubes, soak it in 1 mL of resuscitation solution for resuscitation, and then inoculate it on an agar plate medium for culture. After 12 hours, observe the number of colonies grown on the agar plate medium, which is the number of live bacteria in the sample; then calculate the number of live bacteria in each strain tube after resuscitation based on the dilution multiple and the proportion of the sampling volume.

[0097] Resuscitation live bacteria ratio:

[0098] The resuscitation live bacteria ratio of each strain tube was calculated based on the live bacteria count obtained by measuring the live bacteria count of each strain tube and the total bacteria count obtained by measuring the total bacteria count, according to the formula: resuscitation live bacteria ratio = (live bacteria count / total bacteria count) × 100%.

[0099] In this test, three groups of samples were taken and the above tests and calculations were performed separately to eliminate errors caused by sampling and other factors. The test results are shown in Table 2 below.

[0100] Table 2 Test results of resuscitated live bacteria ratios in Examples 1 to 8 and Comparative Examples

[0101] Sample group 1 ratio (%) Sample group 2 ratio (%) Sample group 3 ratio (%) Example 1 59.12 59.87 60.26 Example 2 60.79 60.41 60.92 Example 3 62.28 62.02 61.96 Example 4 63.87 64.02 63.93 Example 5 63.98 64.06 64.11 Example 6 64.42 64.21 64.73 Example 7 66.79 66.68 67.13 Example 8 66.74 67.02 66.89 Comparative Example 44.72 46.54 45.96

[0102] It can be seen from the data in Table 2 that, compared with the frozen Haemophilus influenzae type b strains of the comparative example, the frozen Haemophilus influenzae type b strains prepared in Examples 1 to 8 of the present application have a significantly improved ratio of revived live bacteria due to the use of a specific preparation process and a specific freezing solution ratio, which also confirms the test results in Table 1.

[0103] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A freeze-drying preparation method for Haemophilus influenzae type b, characterized in that: The following steps are involved: S1. Dissolve the frozen strain of Haemophilus influenzae type b with physiological saline, then place the dissolved bacterial solution in chocolate blood agar medium, and culture it in a CO2 incubator at 35-37°C for 16-24h to obtain a resuscitation strain culture medium; S2, rinsing the bacterial lawn on the surface of the resuscitation strain culture medium obtained in step S1 with physiological saline, inoculating the rinsed bacterial solution into a new chocolate blood agar culture medium, maintaining the temperature at 35-37° C., and culturing in a CO2 incubator for 10-20 hours to obtain a proliferation culture medium; S3, washing the bacterial lawn on the surface of the culture medium for proliferating bacteria obtained in step S2 with a skim milk solution, removing the bacterial solution obtained by washing, shaking it well, and then dispensing it into bacterial tubes to obtain bacterial tubes for dispensing bacterial strains; S4, adding freezing solution to the culture tube of the subpackaged culture obtained in step S3, shaking well, sealing the culture tube with absorbent cotton, cooling by program at -80°C, and then sealing the tube mouth of the culture tube to obtain freeze-dried Haemophilus influenzae type b strains, and freezing and storing; In step S4, the mass fraction ratio of each component of the freezing solution includes: DMEM / F12 culture medium 40-50%, trehalose 6-10%, dimethyl sulfoxide 12-16%, glucose 6-8%, lecithin 2-4%, and the balance is fetal bovine serum.

2. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In step S1 and step S2, the concentration ratio of each component of the chocolate blood agar culture medium includes: 20-25 g / L bovine casein peptone, 15-20 g / L meat peptone, 40-60 g / L corn starch, 3-5 g / L sodium chloride, 14-18 g / L agar, 400-500 mg / L multivitamins, and 80-100 g / L defibrinated bovine blood.

3. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In the step S1, the dissolution with physiological saline is carried out by adding physiological saline and then repeatedly blowing and dissolving.

4. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In the step S1, the cells are cultured in a CO2 incubator, and the concentration of CO2 in the incubator is 4.5-5.5%.

5. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In step S2, the culture is carried out in a CO2 incubator, and the concentration of CO2 in the incubator is 4.5-5.5%.

6. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In step S3, during subpackaging, the subpackaging volume per tube is controlled to be 0.2-0.4 mL.

7. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In step S4, the opening of the culture tube is sealed by hot-melt sealing.

8. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In step S4, the cryopreservation is performed by cryopreservation in liquid nitrogen.

9. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: After step S1, the bacterial moss in the culture medium of the revived strain needs to be sampled and identified before proceeding to step S2.

10. The freeze-drying preparation method of Haemophilus influenzae type b according to claim 1, characterized in that: In the step S2, after that, the bacterial moss in the culture medium of the revived strain needs to be sampled and identified before proceeding to step S3.