Vaccine for preventing and treating diseases caused by Acinetobacter baumannii and preparation method thereof

A technology for Acinetobacter baumannii and vaccine, applied in the biological field, can solve the problems of low yield, difficulty in purification and quality control, poor product uniformity and the like

Active Publication Date: 2019-03-05
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are two main methods for the production of polysaccharide-protein conjugated vaccines. Currently, chemical methods are mostly used in the market. Chemical methods refer to the covalent linkage of CPS or O-PS to carrier proteins through chemical methods. However, the quality control of chemical methods is difficult. Random cross-linking of proteins leads to poor product uniformity, difficulty in purification and quality control, and many steps in the production process, low yield and high cost

Method used

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  • Vaccine for preventing and treating diseases caused by Acinetobacter baumannii and preparation method thereof
  • Vaccine for preventing and treating diseases caused by Acinetobacter baumannii and preparation method thereof
  • Vaccine for preventing and treating diseases caused by Acinetobacter baumannii and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1, the construction of the recombinant vector expressing PglL and rCTB4573

[0085] 1. Construction of recombinant vector expressing PglL

[0086] The amino acid sequence of Neisseria meningitidis glycosyltransferase PglL is shown in SEQ ID No.3, and its coding sequence is shown in nucleotides 180-1994 of SEQ ID No.4. The 1st-6 nucleotides of SEQ ID No.4 are the XbaI recognition site, the 2475-2480th nucleotides are the SacI recognition sequence, the 2487-2492th nucleotides are the PstI recognition site, and the 2510-2492th nucleotides are the PstI recognition site. Nucleotide 2515 is the XhoI recognition site. The 105th-2240th nucleotides of SEQ ID No.4 are the sequence of the PglL expression cassette. In the PglL expression cassette, the expression of PglL is initiated by the tac promoter, and the expression cassette is named tacpglL. Wherein, the 105th-133rd nucleotide of SEQ ID No.4 is the sequence of tac promoter, and the 180th-1994th nucleotide of SEQ...

Embodiment 2

[0093] Example 2. Construction of glycoengineered Acinetobacter baumannii and detection of protein glycosylation

[0094] 1. Competent preparation of Acinetobacter baumannii electroporation

[0095]Cultivate Acinetobacter baumannii overnight at 37°C, subculture in low-salt LB liquid medium at a volume ratio of 1:100 and cultivate 50ml of Baumann's (low-salt LB liquid medium (500mL) formula: 5g peptone, 2.5g yeast powder , 2.5g NaCl, the balance is water, pH 7.0), 30 ℃ continue to cultivate to OD 600 When the value is 0.6, bathe in ice for 30 minutes, then centrifuge at 6000r / min and 4°C for 8 minutes to collect the bacteria, wash with high-pressure sterilized 10% glycerol four times, and finally resuspend the bacteria with 300 μL of 10% sterilized glycerol. Acinetobacter baumannii competent cells for electroshock transformation were obtained.

[0096] 2. Expression and glycosylation of fusion protein expression vector pET28tacpglL-tacrCTB4573 in Bowman

[0097] The pET28tac...

Embodiment 3

[0099] Example 3, Purification of Glycoengineering Acinetobacter baumannii Glycoprotein

[0100] 1. Pick the pET28tacpglL-tacrCTB4573 / Ab monoclonal clone of Example 2, inoculate it in 5ml LB liquid medium containing a final concentration of 50 μg / mL kanamycin, culture it overnight at 37°C, and pass on to LB liquid medium, cultured to OD at 37°C 600 At about 0.6, add IPTG with a final concentration of 1 mM, and cool down to 30° C. for induction for 12 hours to obtain a protein-inducing culture medium, and centrifuge to obtain protein-inducing cells.

[0101] 2. Sample pretreatment

[0102] Take 10g of the protein-induced bacteria in step 1, add 100ml of A1 solution (20mM pH7.5Tris-HCl, 0.5M NaCl, 10mM imidazole, adjust the pH to 7.0), ultrasonically break the bacteria (ultrasound for 4s and pause for 5s, and the cumulative ultrasonic time is 2h), Centrifuge with a centrifugal force of 12 000g to collect the supernatant, and then centrifuge the supernatant again with a centrif...

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Abstract

The invention discloses a vaccine for preventing and treating diseases caused by Acinetobacter baumannii and a preparation method thereof. The vaccine for preventing and treating the diseases caused by the Acinetobacter baumannii provided by the invention has an active component which is an Acinetobacter baumannii glycoprotein obtained through glycosylation of a protein composed of bases from site20 to site 156 of SEQ ID No. 1 or bases from site 1 to site 156 of SEQ ID No. 1. According to the invention, Acinetobacter baumannii glycoprotein modified rCTB4573 prepared by a genetic engineering method is used for preparation of an Acinetobacter baumannii glycoprotein protein conjugate vaccine; uniformity of the vaccine can be improved; production efficiency of the vaccine is improved; the cost is reduced; extensive application prospect is achieved; and the vaccine can be used for preventing the diseases caused by pathogenic Acinetobacter baumannii.

Description

technical field [0001] The invention relates to a vaccine for preventing and treating diseases caused by Acinetobacter baumannii in the field of biotechnology and a preparation method thereof. Background technique [0002] Acinetobacter baumannii (Ab) is an aerobic, non-fermenting, Gram-negative coccobacillus that is ubiquitous in nature, and it has strong viability, no need for special nutrition, high colonization rate, sticky Strong attachment ability, etc., can cause acquired pneumonia, bloodstream infection, abdominal infection, meningitis, central nervous system infection, urinary system infection, skin and soft tissue infection, etc. Ab has been prevalent worldwide and is the most important opportunistic pathogen in nosocomial infections. It can cause explosive epidemic infections in hospitals and is easy to colonize the skin surface, oral cavity, respiratory tract, gastrointestinal tract and urinary tract of hospitalized patients. Risk factors for Ab infection includ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/104A61P31/04C12N15/74C12N15/62C12N15/54
CPCA61K39/104C07K14/212C07K2319/02C07K2319/21C12N9/1048C12N15/62C12N15/74
Inventor 王恒樑刘志成潘超朱力吴军冯尔玲刘先凯孙鹏王东澍曾明王斌井申荣
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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