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Acinetobacter baumannii zinc dependent oligopeptide A1S-1610 recombinant protein and preparation method and application thereof

A technology of Acinetobacter baumannii and protein, applied in the biological field, can solve the problems of toxic and side effects, large toxic and side effects, complex vaccine components of the outer membrane complex, etc.

Active Publication Date: 2015-08-26
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the whole bacteria vaccine is a traditional vaccine, the vaccine components contained in it are complex and have certain toxic and side effects, so the safety is not high
The composition of the outer membrane complex vaccine is complex, and it also contains a large amount of endotoxin, which has great toxic and side effects on the body. Therefore, it is an inevitable trend to develop a quality-controllable, safe and effective Acinetobacter baumannii vaccine

Method used

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  • Acinetobacter baumannii zinc dependent oligopeptide A1S-1610 recombinant protein and preparation method and application thereof
  • Acinetobacter baumannii zinc dependent oligopeptide A1S-1610 recombinant protein and preparation method and application thereof
  • Acinetobacter baumannii zinc dependent oligopeptide A1S-1610 recombinant protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: Cloning of Acinetobacter baumannii A1S_1610 protein

[0059] 1. Firstly, according to the gene sequence of Acinetobacter baumannii 17978 standard strain A1S_1610, the signal peptide analysis software was used to analyze the results. Image 6 .

[0060] 2. According to the analysis results, use the PCR method to amplify the gene fragment of A1S_1610 protein with the whole genome of Acinetobacter baumannii 17978 as a template. The amplification steps are as follows:

[0061] 1) Design the PCR primers as follows, which are SEQ ID NO.7-8 (the base sequence of the restriction site is underlined)

[0062] Forward primer PA1S1610B1 (SEQ ID NO.7):

[0063] 5'-CGC GGATCC GAAGCGACGCGTGCGACA-3'

[0064] BamH Ⅰ

[0065] Reverse primer PA1S1610N2 (SEQ ID NO.8):

[0066] 5'-TTAT GCGGCCGC CTTATTTAACCCGTTGTCCGGTAA-3'

[0067] Not Ⅰ

[0068] In this example, the DNA sequence SEQ ID NO.2 encoding the amino acid sequence of the A1S_1610 protein shown in SEQ ID NO.1 ...

Embodiment 2

[0098] Example 2: Acinetobacter baumannii-17978A1S_1610 protein induced expression, purification and identification of expression form in prokaryotic expression system-Escherichia coli

[0099] 1. Induced expression of target protein

[0100] 1) Take 100 μL of the pGEX-6P-2-A1S_1610 / XL-1blue bacterial solution that was correctly identified by double enzyme digestion and add it to 10 mL of Amp-resistant TB medium, cultivate overnight at 100 rpm at 37°C, take 2 ml of the overnight cultured bacterial solution and add it to 18 mL In the Amp-resistant TB medium (the rest of the bacterial solution is stored in a 4°C refrigerator for later use), culture at 37°C for 2 to 3 hours at a rotation speed of 250rpm, and after secondary activation until the OD600 is 0.8 to 1.2, add 4.4 μL of IPTG to make it The final concentration was 200 μM, and then placed on a shaker to induce expression at 30°C for 3h, at 25°C for 5h, and at 16°C overnight to induce expression.

[0101] 2) Take out the b...

Embodiment 3

[0106] Embodiment 3: Preparation of A1S_1610 protein antigen

[0107] 1. Amplify culture to obtain protein

[0108] Inoculate the pGEX-6P-2-A1S_1610 / XL-1blue bacteria stored in the -80°C refrigerator on the LB ampicillin-resistant plate, and cultivate overnight at 37°C; pick a single colony and inoculate it in 100ml LB ampicillin-resistant medium, 37 Cultivate overnight at 200 rpm; add 100ml of the activated bacterial solution to 2L LB medium containing Amp resistance for secondary activation, culture at 37°C for 3-4 hours until the OD600 is 1.2, add 420ml of IPTG (final concentration is 200uM) After induction in a shaker at 30°C for 3 hours, centrifuge at 6000rpm for 5 minutes to collect the bacteria, add 80ml of PBS to resuspend the bacteria, then ultrasonically lyse the bacteria for 30 minutes, collect the supernatant and 4ml of glutathione-agarose by centrifugation as above Gel 4B binding; a large amount of A1S_1610 fusion protein containing GST tag was obtained.

[0109...

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Abstract

The invention relates to an acinetobacter baumannii A1S-1610 protein, a carrier, engineering bacteria, a composition or a kit which comprises the acinetobacter baumannii 1 A1S-1610 protein, and a preparation method and application of the acinetobacter baumannii 1 A1S-1610 protein, the acinetobacter baumannii 1 A1S-1610 protein is never used in the field of recombinant subunit vaccines, the acinetobacter baumannii 1 A1S-1610 protein can effectively stimulate the body to cause a protective immune response so as to resist acinetobacter baumannii lethal infection. The present invention also discloses a method for preparation of the recombinant protein by construction of an expression vector of the recombinant protein, and transformation of host bacteria, and use of the recombinant protein in the preparation of an acinetobacter baumannii resistant subunit vaccine and a related detection kit. The protective antigen composition A1S_1610 protein is expressed by use genetic engineering technology to clone, and the A1S_1610 protein is high in expression amount, easy to separate and purify, efficient and safe, and the genetic engineering recombinant subunit vaccine has good acinetobacter baumannii infection resistant immune protection effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a protein of Acinetobacter baumannii A1S_1610 and its application in the preparation of Acinetobacter baumannii vaccine. Background technique [0002] Acinetobacter baumannii (Acinetobacter baumannii) is a non-fermenting gram-negative bacillus, which widely exists in nature and belongs to conditional pathogenic bacteria. Widely present in water and soil in nature, hospital environment and human skin, respiratory tract, digestive tract and genitourinary tract, it is an opportunistic pathogen. The distribution of departments is the most in the ICU, followed by patients in the Department of Respiratory Medicine. Most of the infected patients are elderly patients, patients with critical illness and weak body resistance, and patients who have used various invasive operations and long-term treatment with broad-spectrum antibiotics. Can cause pneumonia, burn infection, wound infection, meni...

Claims

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Application Information

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IPC IPC(8): C07K14/22C07K19/00C12N15/31C12N15/62C12N15/70C12N1/21C12P21/04A61K39/02A61P31/04C12R1/19C12R1/01
CPCA61K39/0208C07K14/22C07K2319/23G01N33/56911
Inventor 曾浩邹全明冯强石云敬海明赵莉群
Owner ARMY MEDICAL UNIV
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