Stenotrophomonas maltophilia outer membrane protein and application thereof

A protein and sequence technology, applied in antibacterial immunoglobulin, application, immunoglobulin, etc., can solve the problem of less antigen research

Inactive Publication Date: 2017-05-31
中国人民解放军第307医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few studies on the protective antigen of Stenotrophomonas maltophilia
Among the three common non-fermenting Gram-negative bacilli, Pseudomonas aeruginosa protective antigen research is the earliest and has the most content, and has made various forms of vaccine preparations such as bacterial inactivated vaccines, compon...

Method used

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  • Stenotrophomonas maltophilia outer membrane protein and application thereof
  • Stenotrophomonas maltophilia outer membrane protein and application thereof
  • Stenotrophomonas maltophilia outer membrane protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1, Expression and Purification of Stenotrophomonas maltophilia Outer Membrane Protein

[0070] 1. Genomic DNA of Stenotrophomonas maltophilia K279a was extracted.

[0071] 2. Using the genomic DNA obtained in step 1 as a template, PCR amplification is performed using primers P1 and P2 to obtain PCR amplification products.

[0072] P1: 5′CG GAATTC CAGGACAGCAGTCCACCGAT-3′;

[0073] P2: 5′-CC CTCGAG TCAGAAGCGGGCACCGAT-3'.

[0074] In P1 and P2, the underline marks the restriction sites of EcoR I and Xho I, respectively.

[0075] 3. Use restriction endonucleases EcoR I and Xho I to double digest the PCR amplified product of step 2, and reclaim the digested product

[0076] 4. Digest the pET-30a(+) vector with restriction endonucleases EcoR I and Xho I to recover a vector backbone of about 5388 bp.

[0077] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant vector pET-30a-Smlt4123. According to the sequencin...

Embodiment 2

[0092] Embodiment 2, preparation of antiserum by recombinant protein ompW immunization mice

[0093] Experimental mice: male BALB / c mice (6-8 weeks).

[0094] 1. The recombinant protein ompW solution obtained in step 9 and the control protein solution obtained in step 10 were quantified and diluted to 100 μg / ml respectively to obtain recombinant protein ompW dilution and control protein dilution.

[0095] 2. The experimental mice were randomly divided into two groups (10 mice in each group).

[0096] On day 0, the groups are grouped as follows:

[0097] Experimental group: Take the pre-immune serum (take blood from the eye socket, let it stand at room temperature for 60 minutes, then centrifuge at 2500rpm for 5 minutes, take the upper layer of serum, which is the pre-immune serum), and then use the recombinant protein ompW antigen emulsion I to immunize the experimental mice (subcutaneously in the abdomen and back). Multi-point injection, 100μL / only).

[0098] Control group...

Embodiment 3

[0107] Embodiment 3, antiserum potency determination

[0108] Serum to be tested: the pre-immune serum obtained in Example 2, the post-immunization serum of the experimental group, and the post-immunization serum of the control group.

[0109] 1. Use PBST buffer to test serum 1:1×10 5 Dilute to obtain the serum dilution to be tested.

[0110] 2. The recombinant protein ompW solution obtained in Example 1 was quantified and diluted to 10 μg / ml, then added to a 96-well plate (100 μl per well), and coated at 4° C. overnight.

[0111] 3. After completing step 2, discard the solution in the well, and wash the plate with 0.5% (volume percentage) PBST solution.

[0112] 4. After completing step 3, add 100 μL of the serum dilution to be tested in step 1 to each well, and incubate at 37°C for 30 minutes.

[0113] 5. After completing step 4, discard the solution in the well, and wash the plate with 0.5% (volume percentage) PBST solution.

[0114] 6. After completing step 5, add 100 ...

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Abstract

The invention discloses a Stenotrophomonas maltophilia outer membrane protein and application thereof. The Smlt4123 protein provided by the invention is any one of the following (a1)-(a3): (a1) protein composed of amino acid sequence disclosed as Sequence 4 in the sequence table; (a2) proteins composed of amino acid sequence of Sequence 4 in the sequence table in the 53rd-240th site from the N terminal; and (a3) Sequence-4-derived protein with the same function as the amino acid sequence disclosed as Sequence 4 subjected to substitution and/or deletion and/or addition of one or more amino acid residues. The Smlt4123 protein has higher immunogenicity. The killing test on the whole blood prepared from the immune serum detects that the protein can lower the bacterium carrying capacity in blood in vitro. The challenge test indicates that the antibody generated by the protein immunization can also lower the bacterium carrying capacity in blood in vivo, has certain protection actions and can be used as a key vaccine candidate molecule.

Description

technical field [0001] The invention relates to an outer membrane protein of Stenotrophomonas maltophilia and its application. Background technique [0002] Stenotrophomonas maltophilia is a non-fermenting Gram-negative bacillus that widely exists in nature and hospital environments, and is an opportunistic pathogen. With the extensive use of broad-spectrum antimicrobials and immunosuppressants and the continuous increase of invasive medical operations, the clinical isolation rate of this bacteria is increasing year by year, and it has become an important pathogen of hospital-acquired infections. According to the 2005-2011 data of China CHINET Bacterial Resistance Surveillance Network, the isolation rate of this bacteria ranks 5th-6th among Gram-negative bacteria and third among non-fermenting bacteria, second only to Pseudomonas aeruginosa and Baumannii In 2011, this bacteria accounted for 4.45% of all Gram-negative bacteria and 11.61% of non-fermenting bacteria. SENTRY g...

Claims

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Application Information

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IPC IPC(8): C07K14/195C07K16/12C12N15/31A61K39/02A61K39/40A61P31/04
CPCA61K39/0208A61K2039/505C07K14/195C07K16/1203
Inventor 李艳许广杨汤雪萍尚学义郭雷静
Owner 中国人民解放军第307医院
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