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Preparation and application of an att fusion protein for preventing Staphylococcus aureus infection

A fusion protein, staphylococcus technology, applied in the field of genetic engineering, can solve problems such as poor efficacy of antibacterial drugs

Active Publication Date: 2019-01-01
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, antibiotics were mainly used for the treatment of Staphylococcus aureus infection, but with the widespread use of antibiotics, a large number of drug-resistant bacteria appeared, making the efficacy of antibacterial drugs poor

Method used

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  • Preparation and application of an att fusion protein for preventing Staphylococcus aureus infection
  • Preparation and application of an att fusion protein for preventing Staphylococcus aureus infection
  • Preparation and application of an att fusion protein for preventing Staphylococcus aureus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] This example illustrates the preparation of ATT fusion protein and corresponding experimental related protein.

[0031] 1. Design and synthesis of primers

[0032] Referring to the published trap gene sequence of Staphylococcus aureus on NCBI, two pairs of PCR primers were designed by using Oligo6.67 and DNAStar software. The upstream and downstream primers for amplifying ATT protein were F1 and R1; the upstream and downstream primers for amplifying TRAP protein were F2, R2. See Table 1 and figure 1 . For further directional cloning, BamH I was introduced at the 5' end of the upstream primer, Hind IIII was introduced at the 3' end of the downstream primer, the underline represents the restriction enzyme site, and the wavy line represents the Linker (GSGSGSGS). The above primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0033] Table 1 PCR primers and Linker design

[0034]

[0035] 2. TRAP-pET32a recombinant bacterial cultu...

Embodiment 2

[0059] This example illustrates the comparison of immune effects of ATT, Trap and eAls proteins.

[0060] 1. Animal immunity and challenge test

[0061] Take 100 healthy, female, SPF C57 / B6 mice of the same age and divide them into S.aureus Newman and S.aureus Wood46 challenge groups. Mice were challenged after immunization with ATT protein, and TRAP protein, eALS protein, eAls protein mixed with TRAP protein (eAls+TRAP) and PBS were used as controls at the same time, 10 mice in each group. Each experimental histone protein was purified at 1 mg / ml, emulsified with Freund's complete adjuvant at a ratio of 1:1, and 200 μl of each mouse was injected into the muscle of the leg; 21 days after the first immunization, the protein was mixed with incomplete Freund's adjuvant with Emulsify at a ratio of 1:1, and inject into leg muscles to strengthen immunity.

[0062] In this experiment, S.aureus Newman and Wood46 standard strains were used for challenge. Take the diluted bacterial s...

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Abstract

The invention relates to ATT fusion protein which is formed by linking 9 amino acids at Candida albicans Als3T cell epitopes before an amino acid sequence of TRAP fusion protein of staphylococcus aureus through Linker. The amino acid sequence of the fusion protein is shown in SEQ ID No.2. The invention further relates to genes for encoding the ATT fusion protein as well as a preparation method and an application of the ATT fusion protein. A mouse is immunized with the ATT fusion protein, immunodetection and a toxicity attack test are performed, and results indicate that the ATT fusion protein can induce a body to generate a stronger immune response and to have a better staphylococcus aureus infection prevention function and is an ideal candidate vaccine antigen for preventing infection of staphylococcus aureus.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to the preparation and application of an ATT fusion protein for preventing Staphylococcus aureus infection. Background technique [0002] Staphylococcus aureus (S.aureus) is a common Gram-positive pathogenic bacterium in nature. It is the most common cause of human hospital and community infections and seriously endangers human health. S. aureus is also the main pathogen of mastitis in dairy cows, causing huge economic losses to the dairy industry. In the past, antibiotics were mainly used to treat Staphylococcus aureus infection, but with the widespread use of antibiotics, a large number of drug-resistant bacteria appeared, making the efficacy of antibacterial drugs poor. There is growing awareness that vaccines are the first choice for preventing S. aureus infection. Studies have shown that in the process of anti-Staphylococcus aureus infection, cellular immune responses, especia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/085A61P31/04C12R1/19
CPCA61K39/00C07K14/31C07K14/40C07K2319/00
Inventor 崔玉东刘伟于立权
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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