Preparation method of bi-combined inactivated vaccine

A dual inactivated vaccine and inactivation technology, applied in the field of vaccines, can solve the problems of scattered virus and difficult virus cultivation, and achieve the effects of convenient immunity, reduced stress response and high titer content

Inactive Publication Date: 2015-04-01
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, inactivated vaccines are mainly used for the prevention and control of these two viral diseases, but PCV2 inactivated vaccines have the reality that the virus is difficult to cultivate, and FMDV inactivated vaccines have the risk of spreading the virus due to incomplete inactivation

Method used

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  • Preparation method of bi-combined inactivated vaccine
  • Preparation method of bi-combined inactivated vaccine
  • Preparation method of bi-combined inactivated vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Porcine circovirus type 2, porcine foot-and-mouth disease VP1 subunit dual inactivated vaccine

[0034] 1 Preparation of poisonous seeds for production

[0035] 1.1 Preparation of PCV2ZJ / C strain: Dilute the virus seeds appropriately with virus diluent (serum-free MEM medium), inoculate them on PK-15 cell culture at a multiplicity of infection (M.O.I.) of 0.01, absorb at 37°C for 30 minutes, add MEM cell maintenance solution with 3% (v / v) calf serum and 1mmol / L D-glucosamine hydrochloride, cultured at 37°C for 5 days, repeated freezing and thawing 3 times, harvested virus, virus titer ≥ 10 7.0 TCID 50 / ml.

[0036] 1.2 Activation of VP1 protein expression strains: Streak the preserved prokaryotic expression strains onto a Kan+ resistant culture plate and culture overnight at 37°C. Pick a single colony and insert it into 10mL liquid LB medium containing Kan+, shake and culture at 220r / min at 37°C for 11-16h, and use it as a strain for inducing expression.

...

Embodiment 2

[0080] Embodiment 2 FMDV VP1 subunit, PCV2 dual inactivated vaccine and the immune effect comparison of using two kinds of vaccines (swine foot-and-mouth disease O type synthetic peptide vaccine (polypeptide 2570+7309) and PCV2 inactivated vaccine) to immunize piglets alone

[0081] 1. Materials

[0082] Pig O-type foot-and-mouth disease VP1 subunit, PCV2 dual inactivated vaccine, select the laboratory product (lot number is 201401) in the embodiment 1 for use; Produced by the company (batch number is XXXX), the virus content is at least 10% before inactivation 7.0 TCID 50 / ml; FMD O-type synthetic peptide vaccine (polypeptide 2570+7309), purchased from Shanghai Shenlian Biological Co., Ltd. (batch number XXXX), containing at least 25ug / ml of FMD synthetic peptides.

[0083] 2. Design of animal experiments

[0084] Select 90 weaned piglets aged 21 to 28 days and divide them into 6 groups, 15 pigs in each group; each pig in the 1st and 2nd groups was injected intramuscularly ...

Embodiment 3

[0090] 1. A method for preparing a double inactivated vaccine, characterized in that it comprises the following steps:

[0091] 1) Take the PCV2ZJ / C strain to infect the host cells for virus amplification to ensure that the virus titer is ≥ 10 7.0 TCID 50 / ml, harvest cytovenom;

[0092] 2) The cell venom obtained in step 1) is clarified and concentrated by using a hollow fiber filter column;

[0093] 3) adding β-propiolactone to inactivate the concentrated virus in step 2) in a stirring state at a ratio of 0.1 to 0.3% (v / v), to obtain the first antigen stock solution;

[0094] 4) The pET28a-FMDV VP1 strain was cultured and amplified, and then 0.1-1 mmol / L IPTG was added to the culture system for induction. The induction time was 4-6 hours, and the induction temperature was 25-37°C. wall, collecting inclusion bodies;

[0095] 5) Use CAPS lysate or urea lysate to resuspend the inclusion bodies obtained in step 4) at a concentration of 15-25 mg / mL, collect the supernatant, a...

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Abstract

The invention provides a preparation method of a bi-combined inactivated vaccine. The prepared bi-combined inactivated vaccine provided by the invention can be used for preventing swine type O foot-and-mouth diseases and porcine circovirus type 2. The preparation method provided by the invention is simple, and the vaccine is high in antigen and is convenient to immunize; compared with graded immunization in the prior art, the immunization times can be reduced, the stress reaction can be reduced, and the immunization cost can be lowered; and secondly, comparisons between the effects of immunizing piglets by using a bi-combined vaccine and a monovalent vaccine show that two antigen components cannot interfere with each other in the same injection of the bi-combined vaccine, and moreover, the immune effect of injecting the bi-combined vaccine on the piglets for a single time is higher than the immune effect of sequentially injecting the two vaccines.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to a preparation method of a dual inactivated vaccine. Background technique [0002] Foot-and-mouth disease (FMD) is an acute and highly contagious animal disease caused by foot-and-mouth disease virus (FMDV) infection. FMDV infects domestic and wild artiodactyls such as cattle, sheep, and pigs. Epidemiological surveys and infection experiments have proved that more than 100 species of animals can be infected with this virus. The clinical features of the disease are fast transmission and wide range of epidemics. Blisters and ulcers occur in the oral mucosa, hooves and breasts of adult animals. Young animals often have high mortality due to myocardial damage. In addition to the direct economic loss caused by the death of animals, the production of meat and milk during the disease period of animals stops, the milk production during lactation decreases, the growth of tolerant animal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/135A61P31/14A61P31/20
Inventor 吕茂杰梁武杨保收李亚杰边程
Owner TIANJIN RINGPU BIO TECH
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