Preparation method using bacterial lysate as inactivated vaccine adjuvant

A technology of inactivated vaccines and lysates, which is applied in the field of preparation of bacterial lysates as inactivated vaccine adjuvants, which can solve the problems of limited use of vaccine adjuvants, poor immune enhancement effect, local irritation, etc., and achieve improved vaccine immunity , The production method is simple and the cost is low

Pending Publication Date: 2022-02-11
ZHANGZHOU PIEN TZE HUANG PHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently commonly used vaccine adjuvants, such as white oil adjuvants, usually cause local irritation, and have the disadvantages of weak immune response induction and poor immune enhancement effect, which limits the use of vaccine adjuvants. Therefore, it is necessary to develop a new generation of high-efficiency, Low toxicity, cheap vaccine adjuvant

Method used

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  • Preparation method using bacterial lysate as inactivated vaccine adjuvant
  • Preparation method using bacterial lysate as inactivated vaccine adjuvant
  • Preparation method using bacterial lysate as inactivated vaccine adjuvant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Adjuvant preparation:

[0044] Corynebacterium pseudotuberculosis and Rhodococcus equi were inoculated in LB solid medium at 37°C for 14 hours, picked a single colony, inoculated in 3ml LB liquid medium on a shaker (220rpm) at 37°C for 12h, and inoculated with 600ml LB at a ratio of 3‰ Liquid culture medium, cultivated at 37°C for 12h-16h, measured OD600 reached about 1.0, centrifuged to collect bacteria, diluted with sterilized normal saline to make the concentration of bacteria reached 8.0×109cfu / mL, directly passed through a high-pressure homogenizer, crushed bacteria, Or after 1:1 mixing, the high-pressure homogenizer continuously crushes three times to obtain the vaccine adjuvant mixed with Corynebacterium pseudotuberculosis, Rhodococcus equi and the two, mix with inactivated Pasteurella 1:1, and obtain the vaccine adjuvant added with Inactivated vaccines for Corynebacterium pseudotuberculosis, Rhodococcus equi and a mixture of both. The morphology of Corynebacter...

Embodiment 2

[0047] Safety evaluation of lysates of Corynebacterium pseudotuberculosis and Rhodococcus equi:

[0048] The three batches of adjuvant prepared in Example 1 were injected into 3 healthy mice in a single dose, 5 times the dose and 10 times the dose, and inoculated into the inner muscle of the hind limb. After 14 days of observation, the body temperature and appetite of the inoculated mice were normal, there was no swelling and inflammation at the inoculation site, and no local and systemic reactions occurred; 14 days later, the mice were killed, and the skin of the injection site was cut to observe the pathological changes of the injection site. There was no abnormality in the subcutaneous area of ​​the injection site Muscle tissue at the inoculation site of the inner muscle of the hind limb was normal, and no inflammatory reaction was seen at the injection site. It shows that it has better safety for mice.

example 3

[0050] The promotion effect of Corynebacterium pseudotuberculosis and Rhodococcus equi lysates on mice immunized with Pasteurella inactivated vaccine:

[0051] The lysate of Corynebacterium pseudotuberculosis, Rhodococcus lysate and the lysate of the two mixtures, and 20% aluminum gel adjuvant were mixed with Pasteurella inactivated vaccine at a ratio of 1:9, and then immunized 4-6 weeks old Kunming 5 mice of each strain, 1ml / mouse. After immunization, the indirect ELISA method established in the laboratory was used to continuously detect the antibody level for 8 weeks, and draw the growth and decline rules of the antibody, as shown in figure 2 shown.

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Abstract

The invention relates to a preparation method using bacterial lysate as inactivated vaccine adjuvant. The preparation method comprises the following steps: step 1, respectively putting corynebacterium pseudotuberculosis and rhodococcus equi into an LB culture medium for culturing at 37 DEG C, and harvesting thalli through a centrifugal machine; step 2, diluting each thallus to 8.0* 10<9> cfu / mL by using sterilized normal saline, and crushing the bacterial liquid by using a high-pressure homogenizer; and step 3, after sterility test, mixing the split products of corynebacterium pseudotuberculosis and rhodococcus equi with the two bacteria inactivated by formaldehyde according to a ratio of 1: 1 to obtain the inactivated vaccine added with the adjuvant. The preparation method has the characteristics of safety, high efficiency, strong immune enhancement capability and low cost, and is suitable for producing various bacterial inactivated vaccines.

Description

technical field [0001] The invention belongs to the field of animal husbandry and veterinary medicine, and in particular relates to a preparation method using bacterial lysate as an inactivated vaccine adjuvant. Background technique [0002] Rhodococcus equi is Actinomycetes, Nocardiaceae, Rhodococcus. The bacterium is a Gram-positive facultative intracellular parasite, the colonies are pink or yellow, rod-shaped spherical, acid-resistant, without spores, and most of them have no flagella, but some strains have pili or appendages. The cell wall of Rhodococcus equi is hydrophobic and surrounded by a polysaccharide capsule. The main components of the cell wall are lipids rich in mycolic acids (Mycolic acids) and lipopolysaccharide, wherein the lipopolysaccharide is trehalose dicarboxylate (Trehalose dimycolate, TDM) and lipoarabinomannans (Lipoarabinomannans, LAM). [0003] Rhodococcus equi can cause host macrophages to experience the translocation of NF-κB, and activate the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61P37/04B04B5/00B01F27/90B01F33/71B01F101/44
CPCA61K39/39A61P37/04B04B5/00A61K2039/55594
Inventor 王兴龙黄志鑫李哲李旭鑫张静雅郑玉清谢宏林黄歆然李慧
Owner ZHANGZHOU PIEN TZE HUANG PHARM
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