Bovine brucellosis polysaccharide conjugate vaccine and application thereof

A vaccine, the technology of Yersinia, applied in the field of biomedicine, can solve the problems of infecting humans, abortion of female animals, and the lack of safe human vaccines, etc., and achieve the effect of safe and efficient production process, easy operation and cultivation

Pending Publication Date: 2021-09-17
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current Brucella vaccines are mainly live attenuated vaccines for animals. This vaccine can cause abortion in female animals and can infect humans. There is no safe human vaccine yet.

Method used

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  • Bovine brucellosis polysaccharide conjugate vaccine and application thereof
  • Bovine brucellosis polysaccharide conjugate vaccine and application thereof
  • Bovine brucellosis polysaccharide conjugate vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, expression vector construction

[0037] 1. Construction of expression vector pET28-tacpglL-tacCTB4573

[0038] 1. Artificially synthesize the DNA molecule shown in SEQ ID No.1. In SEQ ID No.1, the 1st-6th nucleotide from the 5' end is the XbaI recognition site, and the 105th-2240th nucleotide is the sequence of the Neisseria meningitidis glycosyltransferase PglL expression cassette, wherein , the 105th-133rd nucleotide is the sequence of the tac promoter, and the 180th-1994th nucleotide is the coding sequence of Neisseria meningitidis glycosyltransferase PglL (protein shown in coding SEQ ID No.2) , the 2475-2480th nucleotide is the SacI recognition sequence, and the 2486-2491th nucleotide is the XhoI recognition site.

[0039] 2. Digest the DNA molecule shown in SEQ ID No.1 with XbaI and XhoI to obtain a gene fragment; digest the pET28a (+) vector with XbaI and XhoI to obtain a large fragment of the carrier; connect the gene fragment to the large fragmen...

Embodiment 2

[0051] Embodiment 2, the construction of recombinant bacteria

[0052] 1. Preparation of competent cells of Yersinia enterocolitica 52212

[0053] Streak Yersinia enterocolitica 52212 on the BHI solid medium plate, culture in a 25°C incubator for 24 hours, pick a single clone and inoculate it in 5 mL of BHI liquid medium, and culture overnight at 25°C on a shaker at 220rpm / min. Inoculate the bacterial solution 1:100 in 50mL liquid BHI medium, culture at 25°C, 220rpm / min for 4-5h, and make the bacterial solution OD 600 When it reaches about 0.5-0.6, place the bacterial solution on ice for 20 minutes. Collect the cells by centrifugation at 5000×g / min at 4°C for 10 minutes, resuspend the cells in pre-cooled sterilized 10% glycerol, centrifuge again, discard the supernatant, and repeat this step three times. After discarding the supernatant for the last time, resuspend the bacteria with 500 μL of 10% glycerol, aliquot 50 μL / tube to be competent, and store at -80°C.

[0054] 2. ...

Embodiment 3

[0057] Embodiment 3, protein expression, purification and detection

[0058] 1. Protein expression

[0059] Strains to be tested: 52212 / pET28-tacpglL-tacCTB4573, 52212 / pET28-tacCTB4573, Yersinia enterocolitica 52212.

[0060] 1. Inoculate the strains 52212 / pET28-tacpglL-tacCTB4573 and 52212 / pET28-tacCTB4573 to be tested in 5 mL of Kan liquid BHI medium with a final concentration of 50 μg / mL, and inoculate Yersinia enterocolitica 52212 in non-resistant cultured overnight at 25°C and 220rpm / min, and then transferred to 5 mL of Kan liquid BHI medium with a final concentration of 50 μg / mL and non-resistant liquid BHI culture at a volume ratio of 1:100 the next day Base, culture at 25°C, 220rpm / min for 4-5h to make the bacterial solution OD 600 Reach around 0.6-0.8.

[0061] 2. After completing step 1, add 5 μL IPTG to the culture system, continue culturing overnight at 25°C, then take 1.5ml of bacterial culture solution and centrifuge at 5000×g / min for 10 minutes to collect the...

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Abstract

The invention discloses a bovine brucellosis polysaccharide conjugate vaccine and application thereof. An exogenous O-glycosylation system is expressed in O:9 serogroup yersinia enterocolitica, so that O-antigen polysaccharide of the O:9 serogroup yersinia enterocolitica is covalently linked to a substrate protein cholera toxin B subunit, and host bacteria are taken as glycosylation engineering bacteria to synthesize the polysaccharide conjugate vaccine. The polysaccharide structure of the glycoprotein is the same as that of bovine brucellosis, and the glycoprotein is expected to have a protective effect on bovine brucellosis in immune animals. Yersinia enterocolitica of the O:9 serogroup belongs to a secondary biosafety hazard organism and is easier to operate and culture than bovine brucellosis, so that the Yersinia enterocolitica is selected as a host bacterium to avoid large-scale culture of more dangerous bovine brucellosis, and the production process is safer and more efficient.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a bovine brucellosis polysaccharide conjugate vaccine and application thereof. Background technique [0002] Brucellosis is a zoonotic disease that is prevalent in the world and frequently occurs in my country, causing huge economic losses every year. The current Brucella vaccine is mainly an attenuated live vaccine for animals. This vaccine can cause abortion in female animals and can infect humans. There is no safe human vaccine. Brucella bovis, also known as Brucella abortus, is a Level 3 biosafety hazard organism that mainly infects cattle and humans. Contents of the invention [0003] The purpose of the present invention is to provide a bovine brucellosis polysaccharide conjugate vaccine and application thereof. [0004] In the first aspect, the present invention protects the recombinant bacteria, which is obtained by introducing the expression vector into Yersinia enterocolit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/62C12N15/74C07K19/00A61K39/385A61K39/095A61K39/39A61P31/04C12R1/01
CPCC07K14/22C12N15/74C12N9/1048A61K39/385A61K39/39A61K39/095A61P31/04C07K2319/55A61K2039/6037A61K2039/55505
Inventor 王恒樑朱力黄竞潘超吴军孙鹏冯尔玲
Owner ACADEMY OF MILITARY MEDICAL SCI
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