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Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof

A recombinant protein and protein technology, applied in the direction of anti-bacterial immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of complex components of the outer membrane complex, complex components of vaccines, and low safety. Achieve the effect of maintaining spatial conformation and immunogenicity, quality and safety controllable, and simple steps

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

[0003] At present, Acinetobacter baumannii has not entered clinical research at home and abroad. The selection of dominant antigens is very important in the research and development of vaccines. Traditional whole-bacteria vaccines contain complex vaccine components and have certain toxic and side effects, so the safety is not high.
At present, some foreign scholars have conducted preliminary research on the outer membrane complex vaccine of Acinetobacter baumannii, but the outer membrane complex has complex components and contains a large amount of endotoxin, which has great toxic and side effects on the body.

Method used

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  • Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof
  • Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof
  • Acinetobacter baumannii 1 A1S-1969 recombinant protein and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Cloning of Acinetobacter baumannii A1S_1969 protein

[0058] 1. First, according to the gene sequence of Acinetobacter baumannii 17978 standard strain A1S_1969, its amino acid sequence is shown in SEQ ID NO.3. Bioinformatics software was used for structural analysis, and the signal peptide sequence was as follows: Figure 5 As shown, its spatial structure analysis is as follows Image 6 shown.

[0059] 2. According to the analysis results, the PCR method was used to amplify the gene fragments of the two fragments of the A1S_1969 protein with the whole genome of Acinetobacter baumannii 17978 as a template (SEQ ID NO.4), and the amplification steps were as follows:

[0060] 1) Design PCR primers as follows, use forward primer P1A1S1969B1 (SEQ ID NO: 9), reverse primer P1A1S1969N2 (SEQ ID NO: 10) to amplify the first fragment, forward primer P2A1S1969B1 (SEQ ID NO: 11), The reverse primer P2A1S1969N2 (SEQ ID NO: 12) amplifies the second fragment (the base s...

Embodiment 2

[0103] Example 2: Induced expression, purification and identification of expression form of Acinetobacter baumannii-17978 1A1S_1969 protein in prokaryotic expression system-Escherichia coli

[0104] 1. Induced expression of target protein

[0105] 1) Take 100 μL of the correct pGEX-6P-2-1A1S_1969 / XL-1blue bacterial solution identified by double-enzyme digestion, add 10 mL of Amp-resistant TB medium, cultivate at 100rpm at 37°C overnight, and add 2 ml of the overnight cultured bacterial solution to 18 mL In Amp-resistant TB medium (the rest of the bacterial solution should be stored in a refrigerator at 4°C for later use), incubate at 37°C for 2-3 hours, rotate at 250 rpm, and activate it for the second time until the OD600 is 0.8-1.2, then 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 3 hours, and overnight at 16°C to induce expression.

[0106] 2) Take out the bacterial liquid after induction ex...

Embodiment 3

[0112] Example 3: Preparation of 1A1S_1969 protein antigen

[0113] 1. Scale up the culture to obtain protein

[0114] Take the pGEX-6P-2-1A1S_1969 / XL-1blue strain stored in the -80°C refrigerator and inoculate it on the LB ampicillin-resistant plate, and cultivate it overnight at 37°C; pick a single colony and inoculate it in 100 ml of LB ampicillin-resistant medium, 37 Cultivate overnight at 200 rpm; add 100 ml of the activated bacterial solution to 2 L of Amp-resistant LB medium for secondary activation, and culture at 37 °C for 3-4 h until the OD600 is 1.2, then add 420 ml of IPTG (final concentration is 200uM) After induction in a shaker at 30°C for 3 hours, the cells were collected by centrifugation at 6000 rpm for 5 minutes. After adding 80 ml of PBS to resuspend the cells, the bacteria solution was ultrasonically lysed for 30 minutes, and the supernatant and 4 ml of glutathione-agarose were collected by centrifugation as above. Gel 4B binding; large amounts of GST-tag...

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Abstract

The invention relates to a 1 A1S-1969 recombinant protein and a preparation method and application thereof. The recombinant protein comprises 46th to 414th site amino acid sequence of 1 A1S-1969, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 5. The recombinant protein is high in expression amount, easy to separate and purify, efficient and safe, can be directly used with an adjuvant, and is used for the preparation of an acinetobacter baumannii infection resistant subunit vaccine and a related detection kit. Confirmed by animal experiments, the genetic engineering recombinant subunit vaccine has good acinetobacter baumannii infection resistant immune protection effect, lays a foundation for the further study on combined vaccines and multicomponent fusion vaccines, and plays an important role for development and application of prevention and control vaccines and diagnostic kits.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a protein of Acinetobacter baumannii 1A1S_1969 and a preparation method and application thereof. Background technique [0002] Acinetobacter baumannii (Acinetobacter baumannii) is a non-fermenting Gram-negative bacillus, widely present in water and soil in nature, hospital environment and human skin, respiratory tract, digestive tract and genitourinary tract, and is an opportunistic pathogen. 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. Domestic data show that Acinetobacter baumannii accounts for more than 70% of clinically isolated Acinetobacter. The resistance rate of Acinetobacter baumannii to the third and fourth generation cephalosporins has reached 63.0% to 89.9%. The resistance rate to four aminoglycosid...

Claims

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Application Information

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IPC IPC(8): C07K14/22C07K16/12C12N15/31C12N15/70C12N1/21A61K38/16A61P31/04G01N33/569
Inventor 冯强蔡昌芝邹全明曾浩石云敬海明
Owner ARMY MEDICAL UNIV
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