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Staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof

A technology of recombinant protein and staphylococcus, which is applied in the field of biomedicine, can solve the problems of insufficient targeting performance of TRAP protein and low immune level, and achieve the effect of enhancing the level of humoral immune response, reducing the immune dose, and accelerating the popularization of vaccines

Pending Publication Date: 2022-05-06
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The first purpose of the present invention is to provide a recombinant TRAP protein antigen of Staphylococcus aureus targeting the DEC205 receptor of dendritic cells, so as to overcome the problems of insufficient targeting performance and low immune level of the current TRAP protein
[0006] The third purpose of the present invention is to provide a recombinant TRAP protein antigen of Staphylococcus aureus targeting the FC receptor of presenting cells, so as to overcome the problems of insufficient targeting performance and low immune level of the current TRAP protein

Method used

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  • Staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof
  • Staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof
  • Staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This example is the construction and prokaryotic expression of TRAP antigen vector targeting DC

[0043] 1. Alignment of mouse and bovine DEC205 receptor protein structures

[0044] The mouse DEC205 receptor structure and Fc receptor structure, the bovine DEC205 receptor structure and Fc receptor structure were found by consulting the literature and NCBI, and through the Blast function of NCBI (https: / / blast.ncbi.nlm.nih. gov / Blast.cgi), compare the primary structure of proteins online, such as figure 2 , whose homology is 85%; use the online comparison website PROMALS3D (http: ∥prodata.swmed.edu / promals3d / promals3d.php), compare the secondary structure of the protein, such as image 3 , their protein folding methods are highly similar; use the RCSBPDB database (http: / / www.rcsb.org / ) and PyMOLWin software to compare the tertiary structure of proteins, such as Figure 4 , the overlap between the two proteins is very high. Such as Figure 5 It can be seen that the ov...

Embodiment 2

[0057] This example illustrates the optimization of T-DEC205 and T-FcR immune conditions

[0058] 1. Antigen Preparation

[0059] Adjust the concentration of purified pET-32a, TRAP, T-DEC205, and T-FcR proteins to 1 mg / mL, 0.5 mg / mL, 0.1 mg / mL, 0.01 mg / mL, and 0.001 mg / mL, respectively. :1 mixed with complete Freund's adjuvant, fully emulsified with an emulsifier and then set aside at 4°C.

[0060] 2. Immunization of Mice

[0061] Eight-week-old female BALB / c mice were randomly divided into 20 groups, with 4 mice in each group. The pET-32a, TRAP, T-DEC205, and T-FcR protein antigens prepared above were injected into the hind leg muscles to immunize the mice for the first time, and the inoculation dose was 100 μL per mouse.

[0062] 3. Isolation of Mouse Spleen Lymphocytes

[0063] Take 3 mice in each group for spleen isolation, prepare 10 mL splenocyte suspension with 1640 medium, add 1 mL red blood cell lysate to resuspend the cell pellet after centrifugation, discard the...

Embodiment 3

[0084] This example illustrates the immune effect of targeting recombinant proteins.

[0085] 1. Immunization of experimental animals

[0086] One hundred 8-week-old female BALB / c mice were randomly divided into four groups. After diluting the protein and mixing it with complete Freund's adjuvant at a volume ratio of 1:1, the mice were first immunized by intramuscular injection in the hind legs, and the inoculation dose was 100 μL per mouse. Three weeks after the initial immunization, the same protein was mixed with incomplete Freund's adjuvant at a volume ratio of 1:1, and the mice were immunized for the second time in the same immunization method. The tagged protein expressed by pET-32a plasmid was mixed with Freund's adjuvant in an equal volume to emulsify and immunize mice as a control.

[0087] 2. Mice challenge

[0088] One week after the booster immunization, different doses of Newman bacteria suspension were used to prechallenge the same batch of unimmunized mice by...

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Abstract

The invention relates to a staphylococcus aureus TRAP recombinant protein antigen of a targeted dendritic cell DEC205 receptor. The nucleotide sequence of the staphylococcus aureus TRAP recombinant protein antigen is shown as SEQ ID No. 1. The invention further relates to a staphylococcus aureus TRAP recombinant protein antigen of the targeted presentation cell FC receptor, and the nucleotide sequence of the staphylococcus aureus TRAP recombinant protein antigen is shown as SEQ ID No.2. The TRAP protein and the receptor peptide fragment of the targeted antigen presenting cell are recombined together, so that the humoral immune response level of a mouse body is enhanced, the cellular immune response level of the mouse is obviously enhanced, and the problem of intracellular infection parasitism of staphylococcus aureus is solved; according to the method, the immunometering of the vaccine is remarkably reduced, the utilization efficiency of the vaccine is improved, and the immune protection rate of the vaccine is increased to 90% and is remarkably higher than that of untargeted TRAP protein, so that the immune cost is reduced, and the popularization of the vaccine for preventing the cow mastitis is accelerated.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a recombinant protein, in particular to a targeted recombinant protein capable of enhancing cellular immune response and anti-infection. Background technique [0002] Studies have proved that S.aureus can not only survive outside the cell, but also infect cells and survive inside macrophages. Prevention requires both a good humoral immune response and a strong cellular immune response. Studies have proved that the antigen linked by the targeting properties of ligands can not only enhance the level of humoral immune response in vivo, but also significantly enhance the level of cellular immune response. [0003] TRAP protein is expressed in all S.aureus, and no homologous protein of TRAP has been found except in S.aureus, and TRAP protein has good immunogenicity, so TRAP can be used as a general anti-staphylococcal vaccine epitope . Dendritic cells are the only antigen-presenting cells th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/385A61K39/085A61P31/04
CPCC07K14/31C07K16/2851A61K39/085A61K39/385A61P31/04C07K2317/622C07K2319/30A61K2039/6056
Inventor 佟春玉宋博翠韩先乐崔玉东梁益民
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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