A kind of Haemophilus parasuis subunit vaccine and its preparation method

A technology of Haemophilus suis and subunit vaccine, which is applied in the application field of preventing Haemophilus parasuis disease, can solve the problems of weak antigenicity, granuloma, and unsatisfactory use effect of Haemophilus parasuis vaccine, and saves money The effect of manpower and time, ease of use

Inactive Publication Date: 2019-07-19
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of Haemophilus parasuis subunit vaccine and preparation method thereof, overcome the defect of prior art, promptly the antigenicity of existing Haemophilus parasuis subunit vaccine is weak, use traditional subunit vaccine adjuvant Agents such as aluminum adjuvant, mineral oil adjuvant, etc. are prone to side effects such as inflammation and granuloma, so the use effect of the existing Haemophilus parasuis vaccine is not ideal.

Method used

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  • A kind of Haemophilus parasuis subunit vaccine and its preparation method
  • A kind of Haemophilus parasuis subunit vaccine and its preparation method
  • A kind of Haemophilus parasuis subunit vaccine and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment one H. parasuis Cloning of omp16 gene and its expression in Escherichia coli BL21(DE3)

[0033] 1. Primer design and synthesis Through the bioinformatics analysis of omp16, a pair of omp16 amplification primers were designed, and the signal peptide part, that is, the N-terminal 8 amino acid sequences were removed; the length of the primer amplification fragment was 162 amino acids and a total of 486bp; -up 5'-cgg gaattc gcgcctgtaggtgaaac -3' (the underlined part is Eco RI restriction site), omp16-dn 5’- ccc aagctt ttagaaacgataggac -3' (the underlined part is Hind III restriction site) primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0034] , H. parasuis The recovery and extraction of the genome take the freeze-dried H. parasuis Bacteria, streak inoculated on TSA solid plate, set at 37°C, 5% CO 2Incubated overnight. On the second day, single clones of activated bacteria were picked and inoculated in TSB medium, and cultur...

Embodiment 2

[0049] Example 2 Preparation of Haemophilus parasuis Omp16 subunit microsphere vaccine

[0050] 1. Preparation of microsphere vaccine by emulsification-ion cross-linking method. Dissolve the purified and renatured Omp16 recombinant protein in 8.8ml of PBS with pH 8.0 to prepare a solution with a concentration of 200 mg / ml, mix it with 10 ml of 2% sodium alginate solution, add 1.2 ml of Tween 80, and fully Stir to combine. Add 28.8 ml of olive oil and 1.2 ml of Span-80 to the mixture, stir at 1500r / min for 20 min, and fully emulsify; add the emulsified solution dropwise to 100 ml of 8% CaCl 2 In the solution, stir at 1200 r / min for 30 minutes to cross-link and solidify; transfer the solidified microsphere suspension to a centrifuge tube, centrifuge at 1500g for 10 min, discard the upper liquid, and use 10ml 0.1 mol / L sodium acetate buffer for microsphere precipitation (pH4.5) washed 3 times, suspended with the same buffer; take 5ml of microsphere suspension into a beaker, add...

Embodiment 3

[0053] Example 3 Efficacy Test of Haemophilus parasuis Subunit Vaccine

[0054] 1. Mouse immune test. Fifty 8- to 10-week-old clean Kunming mice (purchased from the Animal Experiment Center of Fujian Medical University) were divided into 5 groups, 10 in each group. The immunization procedures of each group are shown in Table 2. The injection group was immunized twice with an interval of 14 days; the oral gavage group was immunized three times with an interval of 14 days.

[0055]

[0056] Fourteen days after the last immunization, the orbital venous blood of the mice was collected, the serum was separated, and stored at -20°C for future use.

[0057] Serum-specific antibody detection. Use the purified and refolded Omp16 protein of the present invention to coat the ELISA plate to establish detection H. parasuis Antibody indirect ELISA method. Omp16 protein was diluted with coating solution (0.05 mol / L carbonate buffer, pH 9.6), added to the ELISA plate (200 ng / 50 μl / we...

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Abstract

The invention discloses a subunit vaccine for preventing Glasser's disease and a preparation method thereof. According to a prokaryotic expression method, an outer membrane protein Omp16 with immunogenicity is expressed by cloning a haemophilus parasuis (Haemophilus parasuis, H. parasuis) strain (culture collection number: CGMCC NO. 11145, patent application number for invention: CN105524857A). The haemophilus parasuis subunit microsphere vaccine is prepared by taking the protein Omp16 as an antigen and coating the protein Omp16 by taking chitosan, olive oil and sodium alga acid as materials. For mice subjected to injection and oral immunization by using the vaccine, the immune protective rates of H. parasuis serum type 5 attack are respectively 80% and 60%.

Description

technical field [0001] The invention relates to the technical field of preparation of genetically engineered vaccines for animal infectious diseases. It specifically relates to a method of genetic engineering to obtain outer membrane protein protection antigen from Haemophilus parasuis and make it into a microsphere vaccine and the application of the vaccine in preventing Haemophilus parasuis disease. Background technique [0002] Haemophilus parasuis ( Haemophilus parasuis , H. parasuis ) is an NAD-dependent Gram-negative bacterium belonging to the genus Haemophilus in the family Pasteurellaceae. As an opportunistic pathogen, H. parasuis Under conditions such as pig emergency and immunosuppression, it can infect pigs aged 2-8 weeks, causing Glasser's disease characterized by multiple serositis, meningitis, arthritis, etc. The incidence of the disease is usually 10%-20%, and the mortality rate can reach more than 50%. Porcine Graves disease widely exists in pig farms ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/102A61K39/39A61P31/04
CPCA61K39/102A61K39/39A61K2039/552A61K2039/55511A61K2039/55588
Inventor 郑新添杨小燕李晓华戴爱玲郭霄峰
Owner SOUTH CHINA AGRI UNIV
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