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Swine streptococcicosis-Glasser's disease bivalent subunit vaccine and preparation method thereof

A technology for Haemophilus suis and subunit vaccines, which can be applied to vaccines, multivalent vaccines, and veterinary vaccines, and can solve problems such as poor cross-protection effects

Pending Publication Date: 2019-10-15
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

The effect of cross-protection between strains of different serotypes is not good

Method used

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  • Swine streptococcicosis-Glasser's disease bivalent subunit vaccine and preparation method thereof
  • Swine streptococcicosis-Glasser's disease bivalent subunit vaccine and preparation method thereof
  • Swine streptococcicosis-Glasser's disease bivalent subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The screening of embodiment one Streptococcus suis antigenic protein

[0052] 1 Genome extraction of Streptococcus suis

[0053] Streptococcus suis 05ZYH33 glycerol bacteria preserved in the laboratory were lined in THA medium (containing 10% horse serum), then placed in a constant temperature incubator at 37°C overnight, and a single colony was picked and transferred to THB medium (containing 10% horse serum). % horse serum) in an incubator for static culture, and then expand the culture to 10mL THB medium (containing 10% horse serum), and use the bacterial genome extraction kit to extract the bacterial genome according to the instructions. The extracted genome was stored in a -20 refrigerator for future use.

[0054] 2 Construction and verification of recombinant plasmids

[0055]Primers were designed according to the gene sequence of strain 05ZYH33 in GenBank (accession number CP000407.1) (Table 2-1). Using the genome of the standard strain 05ZYH33 as a template, ...

Embodiment 2

[0090] Example 2 Construction and Verification of Surface Display Carriers

[0091] According to the aforementioned experimental results, in this example, two exogenous proteins of Streptococcus suis antigens MRP and SLY were displayed on the surface of E.coli BL21 (DE3) cells, and AfuA, CdtB, and OppA of Haemophilus parasuis were used. , OppA2 displayed on the surface of Escherichia coli and evaluated its immunogenicity through animal experiments.

[0092] 1 Related primer design

[0093] Linker is a 3-time repeated sequence of 5 amino acids GGGGS that separates two protein sequences, and the linkers at both ends need to be complementary to the base sequences at both ends of the protein. The linearized vector used for cloning should use the pMD-28a-INP-CAP-His plasmid as a template for reverse amplification, and the sequences at both ends should also be complementary to the terminal base sequences of the proteins connected on both sides. Based on the HPS gene sequence (NC_011...

Embodiment 3

[0117] Embodiment 3 animal test

[0118] 1 Vaccine preparation

[0119] 1.1 Surface-displayed dual subunit vaccines

[0120] (1) Shaking bacteria: Take the constructed glycerol bacteria displaying the recombinant strain on the surface, draw lines on LB (containing Amp 100 μg / mL) plates, and place them in a 37°C incubator for 8 hours. On the next day, single clones were picked and inoculated in LB (containing Amp 100 μg / mL) liquid medium, cultured at 37°C with shaking at 180 r / min, and then transferred to 500 mL of the corresponding medium at a ratio of 1:100 , shake at 180r / min in a constant temperature shaker at 37°C.

[0121](2) Bacteria collection and inactivation: collect the bacteria under sterile conditions and wash them with PBS for 3 times, then add an appropriate amount of PBS to resuspend, add formaldehyde with a working concentration of 1.5‰ to inactivate the bacteria, and check whether the bacteria are inactivated after 48 hours completely.

[0122] (3) Quantif...

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Abstract

The invention relates to a swine streptococcicosis-Glasser's disease bivalent subunit vaccine and a preparation method thereof. The vaccine includes Haemophilus parasuis antigen proteins AfuA, OppA2,CdtB and OppA, and Streptococcus suis antigens which are MRP and SLY; or includes a Haemophilus parasuis fusion protein AfuA-OppA2 and a fusion protein CdtB-OppA which are two antigens, and Streptococcus suis antigen proteins MRP and SLY which are two antigens. The vaccine can stimulate a strong immune response in mice, has good cross-protection effect on mice conteracting different serotypes of Haemophilus parasuis and Streptococcus suis, and is superior to traditional inactivated vaccines. The subunit vaccine provides basis for development and research of efficient, broad-spectrum, and inexpensive swine streptococcicosis-Glasser's disease vaccines.

Description

technical field [0001] The invention relates to the field of veterinary medicine, in particular to the prevention and treatment of animal diseases, more specifically to a streptococcosis suis-Haemophilus parasuis dual subunit vaccine and a preparation method thereof. Background technique [0002] Streptococcus suis is a bacterial infectious disease caused by Streptococcus suis (SS), which is characterized by clinical symptoms such as meningitis, septicemia, and arthritis in pigs. It has been a major problem that has plagued the global pig industry for a long time . There are many serotypes of Streptococcus suis, among which Streptococcus suis serotype 2 (SS2) is also a zoonotic pathogen. Humans can be infected with the pathogen through specific transmission routes (such as wound exposure, etc.) If it is not timely, it will cause sepsis, meningitis, etc., causing poor prognosis and seriously threatening public health security. In 1998 and 2005, there was an epidemic of str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/116A61K39/09A61K39/102A61P31/04
CPCA61K39/092A61K39/102A61P31/04A61K2039/70A61K2039/552A61K2300/00
Inventor 王春来刘思国李刚
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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