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Protein of Acinetobacter baumannii hypothetical protein a1s_1523 and preparation method and application

A technology of Acinetobacter baumannii and recombinant protein, which is applied in the biological field to achieve the effects of controllable quality, high expression, and good immune protection effect.

Active Publication Date: 2018-06-08
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, a number of studies have proved that: the inactivated whole bacterial outer membrane protein outer membrane protein vesicle and outer membrane have good immunogenicity and antigenicity, and their immune mice can resist the infection and invasion of Acinetobacter baumannii, However, considering safety and other aspects, the outer membrane protein is safer than the whole bacteria. Therefore, the outer membrane protein may be one of the best candidate antigens against Acinetobacter baumannii

Method used

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  • Protein of Acinetobacter baumannii hypothetical protein a1s_1523 and preparation method and application
  • Protein of Acinetobacter baumannii hypothetical protein a1s_1523 and preparation method and application
  • Protein of Acinetobacter baumannii hypothetical protein a1s_1523 and preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0057] Embodiment 1: Cloning of Acinetobacter baumannii A1S_1523 protein

[0058] 1. First, according to the amino acid sequence of Acinetobacter baumannii 17978 standard strain A1S_1523, use bioinformatics software for structural analysis. For the analysis results, see the attached Figure 6 , so as to determine the gene fragment of the A1S_1523 protein that needs to be amplified.

[0059] 2. According to the analysis results, PCR method was used to amplify the gene fragment of A1S_1523 protein with the whole genome of Acinetobacter baumannii 17978 as a template. The amplification steps were as follows:

[0060] 1) Design the PCR primers as follows, which are SEQ ID NO.7-8 (the base sequence of the restriction site is underlined)

[0061] Forward primer PA1S1523B1: SEQ ID NO.7

[0062] 5'-CGC GGATCC GACTATAAAATTGATCCAACACA-3'

[0063] BamHI

[0064] Reverse primer PA1S1523N2: SEQ ID NO.8

[0065] 5'-TTAT GCGGCCGC TTATTTTTTAGCTGCACTAGCC-3'

[0066] Not I

[0067] In ...

Embodiment 2

[0097] Example 2: Acinetobacter baumannii-17978A1S_1523 protein induced expression, purification and expression form identification in prokaryotic expression system-Escherichia coli

[0098] 1. Induced expression of target protein

[0099] 1) Take 100 μL of the two recombinant engineered strains pGEX-6P-2-A1S_1523 / XL-1blue that were correctly identified by double enzyme digestion and add them into 10 mL of Amp-resistant TB medium, and culture them overnight at 100 rpm at 37°C. Add 2ml of the bacterial solution of the above-mentioned Amp-resistant TB medium into 18mL (the rest of the bacterial solution is stored in a 4°C refrigerator for later use), incubate at 37°C for 2-3 hours, rotate at 250rpm, and reactivate until the OD600 is 0.8-1.2 for the second time, then add 4.4 μL of IPTG was added to make the final concentration 200 μM, then placed on a shaker to induce expression at 30°C for 3h, and overnight at 16°C.

[0100] 2) Take out the bacterial solution after induced expr...

Embodiment 3

[0105] Embodiment 3: Preparation of A1S_1523 protein antigen

[0106] 1. Amplify culture to obtain protein

[0107] Take the pGEX-6P-2-A1S_1523 / XL-1blue strains (1#, 2#) stored in the refrigerator at -80°C and inoculate them on LB ampicillin-resistant plates, and culture them overnight at 37°C; pick a single colony and inoculate it in 100ml LB ampicillin resistance medium, 37°C, 200rpm culture overnight; add 100ml of activated bacterial liquid to 2L LB medium containing Amp resistance for secondary activation, culture at 37°C for 3-4h until OD600 is 1.2, add 420μl IPTG (final concentration: 200μM) was placed in a shaker at 30°C for 3 hours, centrifuged at 6000rpm for 5 minutes to collect the bacteria, and then 80ml of PBS was added to resuspend the bacteria, and the bacteria were ultrasonically lysed for 30 minutes, and the supernatant and 4ml of glutathione-sepharose 4B combined; a large amount of A1S_1523 fusion protein containing GST tag was obtained.

[0108] 2. Use the ...

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Abstract

The invention relates to recombinant protein of A1S_1523 as well as a preparation method and application of the recombinant protein. The recombinant protein comprises A1S_1523 mature peptide, and an amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3. The recombinant protein disclosed by the invention is high in expression quantity and convenient to separate and purify, is efficient and safe, can be directly matched with adjuvants for use, and can be used for preparing acinetobacter baumannii infection resistant subunit vaccines and related detection kits; proven by animal experiments, gene engineering recombinant monovalent subunit vaccines have good immune protective effects on acinetobacter baumannii infection resistance; and the recombinant protein can be used for laying a foundation for further researching combined vaccines and multi-subunit fusion vaccines, and can also play important roles in the development and application of prevention and treatment vaccines and diagnostic kits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a hypothetical protein A1S_1523 of Acinetobacter baumannii and its preparation method and application. Background technique [0002] Acinetobacter baumannii (Acinetobacter baumannii) is a common non-fermenting gram-negative bacillus that exists widely in nature and can normally colonize the surface of human skin and in the cavity. It is an opportunistic pathogen that can mainly cause pneumonia, blood Flu infection, skin and soft tissue infection, endocarditis and meningitis, etc. With the increasing detection rate of its drug-resistant strains year by year, Acinetobacter baumannii has become an important pathogen worldwide. Among the strains of Acinetobacter baumannii isolated from various nosocomial infections, multi-drug resistant (MDR) strains accounted for 65%. my country's CHINET antimicrobial resistance monitoring network data show that in 14 teaching hospitals in 10 province...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/22C07K19/00C12N15/31C12N15/62C12N15/70C12N1/21C07K16/12A61K39/02A61P31/04
CPCA61K39/0208C07K14/22C07K16/1217C07K2319/23G01N33/56911
Inventor 邹全明石云曾浩冯强杨赟蔡昌芝
Owner ARMY MEDICAL UNIV
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