Preparation method and application of staphylococcus aureus isdbid-trap fusion protein

A technology of fusion protein and Staphylococcus, which is applied in the field of genetic engineering, can solve the problems of unreported immunogenicity and immune protection of IsdB and TRAP fusion protein, achieve good immunogenicity and immune protection, simplify the preparation process, Improve the effect of protective effect

Active Publication Date: 2011-12-14
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report about the immunogenicity and immune protection of IsdB and TRAP fusion protein

Method used

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  • Preparation method and application of staphylococcus aureus isdbid-trap fusion protein
  • Preparation method and application of staphylococcus aureus isdbid-trap fusion protein
  • Preparation method and application of staphylococcus aureus isdbid-trap fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Staphylococcus aureus IsdB immunodominant fragment (IsdB id ) to select and determine

[0036] 1 Materials and methods

[0037] 1.1 Strains and experimental animals

[0038] S.aureus Newman strain (gifted by Eijkman Winkler Laboratory of University Medical Center Utrecht, Netherlands; Immunogenicity of Staphylococcus aureus agglutinating factor A. Feng Hao, Zhu Zhanbo, Cui Yudong, etc. Acta Biological Engineering, 2009, 25(8): 1180- 1186), S.aureus Wood46 strain (provided by Heilongjiang Bayi Agricultural University; Immunogenicity of Staphylococcus aureus agglutinating factor A. Feng Hao, Zhu Zhanbo, Cui Yudong et al. Acta Biological Engineering, 2009, 25(8): 1180- 1186), HLJ855-23-1 (a strain isolated from cow milk with mastitis disease, provided by Heilongjiang Bayi Agricultural University; immunogenicity of staphylococcus aureus agglutinating factor A. Feng Hao, Zhu Zhanbo, Cui Yudong, etc. Biology Engineering Journal, 2009, 25(8): 1180-1186). The p...

Embodiment 2

[0096] Embodiment 2 Staphylococcus aureus IsdB id -Expression of TRAP fusion protein in Escherichia coli

[0097] 1 material

[0098] Bacterial strains, tool enzymes, main reagents and kits, etc., are the same as in Example 1.

[0099] 2 methods

[0100] 2.1 Design and synthesis of primers

[0101] The IsdB immunodominant fragment (isdB) determined according to the published Trap gene sequence and the above-mentioned embodiment one test selection id ) sequence, using Oligo6.67 and DNAStar software to design two pairs of PCR primers, respectively named as F1, R1 (for cloning isdB id gene fragment) and F2, R2 (for cloning the trap gene fragment), R1 and F2 contain complementary linker (shown in black letters). The isdB3-trap gene fragment was amplified by overlapping extension PCR method, using primers F1 and R2 containing connecting peptides to connect and amplify. A restriction enzyme site BamHI (shown underlined) was introduced at the 5' end of the upstream primer F1, a...

Embodiment 3I

[0127] Embodiment 3IsdB3-Trap, IsdB, Trap protein immune effect comparison

[0128] 1 material

[0129] 1.1 Recombinant protein: purified IsdB id - Trap, IsdB, Trap proteins.

[0130] 1.2 ELISA detection reagents and kits: ELISA plates are products of Corning; Goatanti-mouse IgG-HRP was purchased from Beijing Boaosen Biotechnology Co., Ltd.; goatanti-mouse interleukin 2, 4 and goat anti-mouse γinterferon cytokine ELISA quantitative detection The kit is a product of American R&D Company.

[0131] 1.3 Experimental mice, immune adjuvants and other reagents: same as in Example 1.

[0132] 2 Experimental methods

[0133] 2.1 Immunogen preparation and animal immunization

[0134] Take 228 healthy female mice of 18-20g, and randomly divide them into 5 groups, which are fusion protein IsdB id-Trap immunization group, recombinant protein IsdB immunization group, recombinant protein Trap immunization group, recombinant protein IsdB and Trap mixed immunization group (IsdB+Trap), PB...

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Abstract

The invention provides staphylococcus aureus Iron-regulated surface determinant B immunodominant fragment (IsdBid)-target of RNAIII activating protein (TRAP) fusion protein. The amino acid sequence of the staphylococcus aureus IsdBid-TRAP fusion protein is shown by SEQ ID No. 1. A preparation method for the staphylococcus aureus IsdBid-TRAP fusion protein comprises the following steps of: selecting a staphylococcus aureus IsdB immunodominant fragment (IsdB immunodominant fragment, IsdBid), connecting the IsdBid and a trap gene by using Linker through the overlapping primer extension polymerase chain reaction (PCR) technology, performing pronucleus expression on a gene of the IsdBid-TRAP fusion protein, and purifying the fusion protein. According to the result of detection, the immune effect of a vaccine which is prepared from the IsdBid-TRAP fusion protein and is used for immunizing mice is obviously superior to that of the single immunization of the IsdB and TRAP and the mixed immunization of the IsdB and the TRAP, so the staphylococcus aureus IsdBid-TRAP fusion protein is an ideal candidate antigen for preparing staphylococcus aureus vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a Staphylococcus aureus IsdB id - Preparation method and application of TRAP fusion protein. Background technique [0002] Staphylococcus aureus (S.aureus) is a Gram-positive bacterium, which is not only the main pathogen causing various human infections, but also the main pathogen causing mastitis in cows. However, with the widespread use of antibiotics in the treatment of S.aureus infection clinically, a large number of S.aureus strains have emerged, resulting in little or no effect of antibiotic treatment. Therefore, immunoprophylaxis and immunotherapy against S.aureus infection have become the focus of research. In the past 40 years, people have done a lot of research on S.aureus whole-bacteria inactivated vaccines, capsular polysaccharide-conjugated vaccines, toxoids, bacterial surface adhesins and bacterial structural protein subunit vaccines, but the immune protection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K39/085A61P31/04C12R1/445C12R1/19
Inventor 崔玉东朱战波王宁马金柱迟佳琦
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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