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Haemophilus parasuis fusion protein CdtB-OppA having immunizing protection

A technology of Haemophilus suis and fusion protein is applied in the field of veterinary vaccines, which can solve the problems of difficult diagnosis and prevention of pig diseases, low feed utilization rate, poor growth and development of pigs, etc.

Active Publication Date: 2019-09-13
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the disease shows an obvious upward trend in intensive pig farms, mainly causing death of suckling piglets and weaned piglets, poor growth and development of pigs, low feed utilization rate, and mixed infection of Haemophilus parasuis and other pathogens is very common , the isolation rate of Haemophilus parasuis from clinically ill pigs is as high as 20%, which also brings great difficulties to the diagnosis and prevention of swine diseases (Kim J, HK Chung, T Jung, et al., Postweaning multisystemic wasting syndrome of pigs in Korea:prevalence,microscopic lesions and coexisting microorganisms.Journal of Veterinary Medical Science,2002:64,57)

Method used

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  • Haemophilus parasuis fusion protein CdtB-OppA having immunizing protection
  • Haemophilus parasuis fusion protein CdtB-OppA having immunizing protection
  • Haemophilus parasuis fusion protein CdtB-OppA having immunizing protection

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1, construction of recombinant plasmid pMD-28a-INP-cdtB-oppA-His

[0022] 1. Extraction of Genomic DNA from Haemophilus parasuis (Hps)

[0023] Genomic DNA of Haemophilus parasuis (Hps) was extracted according to the following method.

[0024] Centrifuge 1 mL of the overnight culture of Hps (CVCC 3361) at 12,000 rpm for 1 minute, and discard the supernatant. Add 40 μL DB solution (TIANamp Bacteria DNA Kit, Tiangen Biological Co., Ltd.), 160 μL lysozyme and 8 μL RNaseA to the cell pellet. Shake vigorously to mix well. Incubate at 37°C for 30-60 minutes, and invert the centrifuge tube several times. Add 200 μL of DLT solution (TIANamp Bacteria DNA Kit, Tiangen Biological Co., Ltd.) and 25 μL of proteinase K (TIANamp Bacteria DNA Kit, Tiangen Biological Co., Ltd.), and immediately mix it gently by inversion. Place in a 65°C water bath for at least 30 minutes, and invert the centrifuge tube several times. Centrifuge at 12000rpm for 3-5 minutes, and pipette a...

Embodiment 2

[0058] Example 2, Presentation and expression of Haemophilus parasuis fusion protein CdtB-OppA

[0059] 1. Construction of recombinant expression strains presenting and expressing CdtB-OppA

[0060] The recombinant plasmid pMD-28a-INP-cdtB-oppA-His was transformed into E.coli DH5α competent cells, and then spread on LB (containing Amp 100 μg / mL) plates for resistance screening. The next day, single clones were picked and inoculated into LB (containing Amp 100 μg / mL) liquid medium for shaking culture at 37°C. After 12 hours, the plasmid was extracted and sent to Jilin Kumei Biological Company for sequencing. The correct recombinant expression vector verified by sequencing was transferred into E.coli BL21(DE3) competent cells for resistance screening, single clone was selected for PCR identification, and the positive clone was named BL21(cdtB-oppA).

[0061] 2. Presentation and expression of fusion protein CdtB-OppA

[0062] Take BL21 (cdtB-oppA) single clone, enrich and cultu...

Embodiment 3

[0063] Example 3. Immunoprotective Identification of Fusion Protein CdtB-OppA of Haemophilus parasuis

[0064] 1. Vaccine preparation

[0065] Pick BL21(cdtB-oppA) monoclonal and inoculate it in LB (containing Amp 100μg / mL) liquid medium, culture at 37°C with shaking at 180r / min, then transfer to 500mL corresponding medium at a ratio of 1:100 , shake at 180r / min in a constant temperature shaker at 37°C.

[0066] Harvest bacteria under sterile conditions and wash with PBS 3 times, then add appropriate amount of PBS to resuspend, add formaldehyde with a working concentration of 1.5‰ to inactivate the bacteria, and smear the plate after 48 hours to check whether the inactivation is complete.

[0067] The resuspended bacterial solution was taken for SDS-PAGE and then transferred to a membrane for Western-blot. The commercial mouse His antibody was used as the primary antibody, goat anti-mouse IgG (Dylight 680 labeled) was used as the secondary antibody, and His The labeled prote...

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Abstract

The invention relates to haemophilus parasuis fusion protein CdtB-OppA having immunizing protection. Through experiment, the protection ratio of a fusion protein CdtB-OppA immunity group is 90% whichis higher than that (80%) of a purification protein CdtB+OppA immunity group. In the form of fusion protein, reduction of technology complexity during production and reduction of cost can be facilitated, besides, the immune effects are better, and the haemophilus parasuis fusion protein CdtB-OppA can be separately used or can be combined with other protein together to be used as a united vaccine.

Description

technical field [0001] The invention belongs to the field of veterinary vaccines, in particular to a fusion protein of Haemophilus parasuis with immune protection and a vaccine containing it. Background technique [0002] China has always been the country with the largest number of live pigs in the world, so ensuring the safe production of pigs is related to the national economy and the people's livelihood, and pig vaccines also account for 60-70% of veterinary vaccines. There are many types of swine diseases, among which Haemophilus parasuis (also known as Glaser's disease) is caused by Haemophilus parasuis (HPS) with meningitis, multiple serositis, Bacterial infectious disease characterized by pericarditis and arthritis. In 1910, the German scientist Glasser first isolated and described Haemophilus parasuis in the serous secretion of sick pigs, so the disease was also called Glasser's disease. Haemophilus parasuis is a serious hazard to the pig industry and is widespread...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/102A61P31/04C12R1/19
CPCC07K14/195C12N15/70A61K39/102A61P31/04A61K2039/70Y02A50/30
Inventor 王春来刘思国李刚
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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