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Preparation and application of ATM fusion protein for preventing infection of S. aureus and C. albicans

A technology for staphylococcus infection and Candida albicans, which is applied in the field of preparation of ATM fusion protein, can solve the problems of poor milk quality, emergence of drug-resistant strains, impact on the economic benefits of animal husbandry and dairy industry, etc. Immunogenicity, the effect of enhancing immune protection

Inactive Publication Date: 2020-05-12
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

S.aureus is also one of the important pathogens that cause mastitis in dairy cows, which can lead to a decrease in milk production and poor milk quality, seriously affecting the economic benefits of animal husbandry and dairy industry
In the past, antibiotics were the main treatment for S.aureus infection, but due to the long-term use and abuse of antibiotics, a large number of drug-resistant strains have emerged

Method used

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  • Preparation and application of ATM fusion protein for preventing infection of S. aureus and C. albicans
  • Preparation and application of ATM fusion protein for preventing infection of S. aureus and C. albicans
  • Preparation and application of ATM fusion protein for preventing infection of S. aureus and C. albicans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] This example illustrates the preparation of ATM fusion proteins and related experimental proteins.

[0032] 1. Design and synthesis of primers

[0033] Refer to the S. aureus mntc gene sequence published on NCBI (ID: 12330566), use Primer 5.0 software to design specific upstream and downstream primers, and introduce BamH I at the 5'end of the upstream primer, and HindIII digestion at the 3'end of the downstream primer Site. The primer sequence is as follows: the underlined straight line represents the restriction site, and the wavy line represents Linker-(GS) 4 , The dotted line represents the Als3 T cell epitope. The above primers were all synthesized by Jilin Kume Biotechnology Co., Ltd.

[0034] Table 1 PCR primers and Linker design

[0035]

[0036] 2. MntC-pET28a / BL21(DE3) recombinant bacteria culture and plasmid extraction

[0037] Take the glycerol-preserved MntC-pET28a / BL21(DE3) recombinant bacteria frozen at -70℃, and use the inoculation ring to stain the bacteria + S...

Embodiment 2

[0050] This example illustrates the comparison of the immune effects of ATM, MntC and Als proteins.

[0051] 1. Animal immunity and challenge test

[0052] Thirty healthy, female, SPF-grade Babl / c mice of the same age were divided into ATM experimental group, Als3 and MntC mixed experimental group, MntC experimental group, Als3 experimental group and PBS group, with 6 in each group. Purify each experimental histone 1mg / ml, emulsify it with Freund’s complete adjuvant at a ratio of 1:1, and inject 100μg of mouse into the leg muscle for immunization; 21 days after the first immunization, combine the protein and incomplete Freund’s adjuvant with It is emulsified at a ratio of 1:1, and injected into the leg muscles to strengthen immunity.

[0053] After 14 days of booster immunization, mice were injected with S.aureus Newman (1.5×10 9 CFUs / only) and C.albicans SC5314 (6×10 7 CFUs / only) for challenge, take out the liver, spleen, lung and kidney, place them in 2mL PBS buffer for thorough ...

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Abstract

The invention relates to ATM fusion protein. 9 amino acids of the T-cell epitope of C. albicans Als3 are connected to the front of the amino acid sequence of S. aureus MntC fusion protein through a Linker, and the amino acid sequence of the fusion protein is shown in SEQ ID No. 1. In the invention, the T-cell epitope of Als3 is fused with the MntC protein so as to prepare the recombinant ATM fusion protein, and immunogenicity study is conducted on the ATM fusion protein. It is verified through results that organisms are induced to produce better immune response and resistance to S. aureus infection and a better immune protection effect after the T-cell epitope of Als3 is fused with the MntC protein, and MntC-induced effects of immune-protection and resistance to S. aureus infection are enhanced significantly through the T-cell epitope of Als3; and meanwhile, the ATM fusion protein also has certain effect of resistance to C. albicans infection, and is ideal candidate fusion protein forpreparation of reagents with resistance to S. aureus infection and C. albicans infection, and the fusion protein has better immunogenicity, and is capable of inducing the organisms to produce good effects of immune protection.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and relates to the preparation and application of an ATM fusion protein for preventing the infection of Staphylococcus aureus and Candida albicans. Background technique [0002] Staphylococcus aureus (S. aureus) is a common conditioned pathogen that can be infected by humans and animals. It often colonizes the nasal cavity, skin and digestive tract, and can cause skin and soft tissue infections, sepsis, and heart disease. Many diseases such as meningitis and pneumonia. S. aureus is also one of the important pathogenic bacteria that causes mastitis in dairy cows, which can cause a decline in milk production and poor milk quality, which seriously affects the economic benefits of animal husbandry and dairy industry. In the past, antibiotics were the main treatment for S. aureus infection, but due to the long-term application and abuse of antibiotics, a large number of resistant strains appeared. Ther...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/085A61K39/00A61P31/04A61P31/10
CPCC07K14/31C07K14/40C12N15/70A61K39/0002A61K39/085A61P31/04A61P31/10C07K2319/40
Inventor 崔玉东杨思宇
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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