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Brucella molecule marking and virulence deletion attenuated vaccine and preparation method

A technology of Brucella and attenuated vaccine, applied in the direction of bacterial antigen components, bacteria, antibacterial drugs, etc., can solve the problems of disease, adverse reactions between humans and animals, strong virus, etc., and achieve the effect of wide application and practical value.

Inactive Publication Date: 2008-05-28
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to disclose a kind of Brucella attenuated vaccine ΔS19-1 and ΔS19-2 that can distinguish natural infection or wild virus infection, and solve the problem that conventional Brucella vaccines cannot distinguish whether people and animals are artificially immunized or immunized. Wild bacteria infection, strong virus, easy to cause adverse reactions to inoculated humans and animals, and even the problem of disease

Method used

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  • Brucella molecule marking and virulence deletion attenuated vaccine and preparation method
  • Brucella molecule marking and virulence deletion attenuated vaccine and preparation method
  • Brucella molecule marking and virulence deletion attenuated vaccine and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Construction of homologous recombination vector (suicide plasmid)

[0068] 1 Materials and methods

[0069] 1.1 Bacterial strains, plasmids, vectors Brucella S19 strain, DH5 a, pBluescript SK + Preserved in our laboratory; pBK-CMV was purchased from Stratagene; pSP-Luc NF + Purchased from Promega Company; pIB279 was donated by Dr. Jiang Jiandong of Nanjing Agricultural University; pMD18-T simple vector was purchased from TakaRa Company.

[0070] 1.2 Reagents Restriction endonuclease, T4 DNA ligase, DNA polymerase I (Klenow large fragment), Escherichia coli DNA polymerase I, LA-Taq DNA polymerase, 1kbp DNA Ladder Marker, plasmid DNA extraction kit, DNA gel The gel recovery kits were all products of TaKaRa Company; the luciferase detection system was purchased from Promega Company.

[0071] 1.3 PCR amplification

[0072] 1.3.1 Amplification containing the Bp26 gene homologous recombination guide sequence According to the sequence of the Bp26 gene and the characteristi...

Embodiment 2

[0083] Construction of Brucella Molecular Marker and Virulence Lost Vaccine Strain ΔS19-2

[0084] 1.1 Reagents The luciferase detection system was purchased from Promega, and other reagents were the same as in Example 1.

[0085] 1.2 Liver soup medium Take fresh beef liver, remove fat and fascia, mince it, weigh 500g, add 1000mL of tap water to mix with it, boil in a pot for 1h, filter, add water to make up the original amount, add NaCl5g, peptone 10g. Liver soup agar preparation: Take 1000 mL of the above liver soup medium, add 20 g of agar, and sterilize for later use

[0086] 1.3 Electroconversion

[0087] 1.3.1 Preparation of Competent State Take 240mL pre-logarithmic growth mid-phase (OD 600 =0.15) Put the Brucella liquid culture into a pre-cooled 250mL centrifuge tube, quickly place the culture in the ice-water mixture for 15-30min, shake slowly from time to time to ensure that the contents are fully cooled. Then, centrifuge at 1000×g for 15 minutes at 4° C. to recov...

Embodiment 3

[0101] Identification of ΔS19-2 virulence

[0102] In Example 2 of the present invention, we deleted Bmp18, one of the main virulence genes of S19, and constructed a mutant strain of ΔS19-2. Theoretically, ΔS19-2 is less virulent than S19. In the present invention, mice are used as experimental animals to detect the bacteria content of ΔS19-2 and S19 in the spleen of mice inoculated at different times, and to inoculate mice with different high concentrations of ΔS19-2 and S19, and to observe the clinical symptoms and the minimum lethal number of mice , and compared their virulence.

[0103] 1 Materials and methods

[0104] 1.1 Materials

[0105] 1.1.1 Strains

[0106] See Example 2

[0107] 1.2 Method

[0108] 1.2.1 Bacteria count method

[0109] (1) Wash the 48-hour Brucella culture with sterile saline, and make a 1 billion / mL bacterial suspension using standard turbidimetry.

[0110] (2) Then make 10-fold serial dilutions per mL: 100 million, 10 million, 1 million, 1...

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Abstract

The invention relates to a Brucella vaccine, in particular to the molecular marker and virulence gene deletion of Brucella vaccine strain. The study uses luciferase modified gene (Luc NF plus) to replace the partial fragment of Bp26 gene of Brucella attenuated vaccine S19 strain by constructing suicide plasmid and adopting the method of targeted homologous recombination (gene targeting), so as to damage the expression of the immunogenicity protein BP26 and construct the gene deletion mutant strain Delta S19-1 of the Brucella Bp26. The BMP18 protein is one of the main virulence factors of Brucella. The invention adopts the same method to exclude the Bmp 18 gene of Delta S19-1, so as to lead the Delta S19-1 not to express the Bmp 18 protein and the Brucella virulence gene deletion mutant strain Delta S19-2 is constructed. The invention solves the problems that the conventional Brucella vaccine can not distinguish between the artificial immunization and the wild bacteria infection of people and animals, the virus is strong and the vaccine is easy to cause the illness of inoculated people and animals. The invention has important significance and practical application value of the monitoring, diagnosis, purification and all the controls of Brucella.

Description

technical field [0001] The present invention relates to a kind of brucella attenuated vaccine and production technology, especially provide a kind of brucella attenuated vaccine that can distinguish natural infection or wild virus infection, be used for being able to distinguish human beings and animals whether they are vaccinated or naturally infected; The invention also discloses a production method of the vaccine, which belongs to the technical field of immune vaccine production. Background technique [0002] Brucellosis (Brucellosis) is a zoonotic infectious disease caused by small club-shaped Brucella, which is widely prevalent all over the world. The economic loss caused by this is as high as billions of dollars every year, and the disease also poses a serious threat to human health. Since the 1990s, the incidence of human and animal diseases has been on the rise again. [0003] There are many types of Brucella vaccines currently available. Recombinant protein vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/10A61P31/04C12N1/21C12N15/09
Inventor 闫广谋王兴龙
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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