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Staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein as well as preparation method and application thereof

A technology of fusion protein and Staphylococcus, which is applied in the fields of molecular biology and genetic engineering, can solve the problems of limited immune protection, achieve the effect of preventing the preparation process, facilitating in vitro expression, and improving the protective effect

Active Publication Date: 2014-02-12
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 Staphylococcus aureus ITC fusion protein, which expresses multiple antigens of Staphylococcus aureus in tandem, overcomes the existing single antigen molecule as an immunogen for immunization, and the immune protection provided by it problem of limited effect

Method used

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  • Staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein as well as preparation method and application thereof
  • Staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein as well as preparation method and application thereof
  • Staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein as well as preparation method and application thereof

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Experimental program
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Embodiment 1

[0103] This embodiment is used to illustrate the immunodominant fragment of Staphylococcus aureus ClfA (ClfA N3 ) selection and OK.

[0104] (1) Bioinformatics analysis of segmental expression of ClfA

[0105] Enter the PDB protein database (http: / / www.rcsb.org / pdb / home / home.do), download the tertiary structure of ClfA, and use PyMOL software for analysis. The A region of ClfA is divided into three structural domains, namely N1, N2, and N3 domains, wherein the N2N3 fragment has binding activity to neutralizing antibodies and other biological activities, while N1 does not have these activities. Therefore, this experiment mainly focused on ClfA N2N3 fragments for analysis and research. According to bioinformatics analysis, truncation between N2 and N3 will not affect or destroy ClfA N2N3 Spatial conformation, truncation between the two domains N2 and N3 does not disrupt possible T-cell epitopes, nor does it disrupt possible B-cell epitopes since the truncation site is buried...

Embodiment 2

[0155] This example is used to illustrate the expression of ITC fusion protein and its immune protection detection.

[0156] (1) Design and synthesis of primers

[0157] PCR primers were designed according to the published gene sequence of Staphylococcus aureus Newman strain (GenBank Accessed Number NC_009641.1), and the primers were all in the 5'-3' direction. The underlined sequence in the primer is the homology arm sequence and termination sequence of the expression vector pET30a-TEV-LIC to be connected, the bold sequence is the flexible linker connecting the two genes, the first linker1 is (GS) 4 sequence, the second linker2 is (G4S)2 sequence, and the nucleotide sequence of the primer is shown in Table 5. The sequence of isdB (removing the amino acid sequence encoding the 1-40 position and three stop codons, the full length of the isdB sequence is 1,815bp) is shown in SEQ ID NO.10, isdB id The sequence of traP (position 376-1,083 from the isdB sequence, a total of 708bp...

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Abstract

The invention discloses a staphylococcus aureus ITC (Inverse Transition Cycling) fusion protein which is a protein formed by fusing IsdB immunodominance fragments (IsdBid) and Trap as well as a ClfA immunodominance fragment N3 area (ClfAN3); an amino acid sequence of the fusion protein is as shown in SEQ ID NO.2. The invention further provides a coding gene, a preparation method and application of the fusion protein in preparing a vaccine for preventing staphylococcus aureus infection. The ITC fusion protein disclosed by the invention contains various antigens, and therefore, immune effect is good, protective effect of preventing staphylococcus aureus infection is improved; compared with the immune result of any of ClfA, IsdB and TraP, the ITC fusion protein immune effect is better than that of single protein; moreover, the ITC contains Trap with smaller molecular weight and has an immunodominance fragment with two antigens, so that peptide chain length is not lengthy, and therefore, the fusion protein is beneficial to in-vitro expression and has important value in developing and applying a novel vaccine for preventing staphylococcus aureus.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and relates to a Staphylococcus aureus ITC fusion protein and its preparation method and application, namely, the ITC fusion protein formed by fusing the IsdB immunodominant fragment with the N3 region of the Trap and ClfA immunodominant fragment and its preparation methods and applications. 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, the immunoprevention and immunotherapy of S.aureus infection have become the focus of current research. In the past 40 years, people have done a lot of rese...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/085A61P31/04
Inventor 崔玉东朱战波于立权陈龙欣李闰婷
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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