Preparation method and application of staphylococcus aureus TAF fusion protein

A Staphylococcus, fusion protein technology, applied in the field of genetic engineering, can solve the problems of FnBPA, Trap and Hla fusion protein immunogenicity and immune protection, etc., to achieve good immunogenicity and immune protection, good immunity Protective effect, effect of enhancing protective effect

Active Publication Date: 2016-03-09
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there are no reports on the immunogenicity a...

Method used

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  • Preparation method and application of staphylococcus aureus TAF fusion protein
  • Preparation method and application of staphylococcus aureus TAF fusion protein
  • Preparation method and application of staphylococcus aureus TAF fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example illustrates S. aureus FnBPA immunodominant fragment (FL) selection and determination.

[0039] 1. Referring to the published FnBPA gene sequence, the data obtained from the study and the epitope analysis of the online software, the gene sequence was divided into two segments, and the 301-430 amino acids in the N region of the FnBPA protein were named as FnBPA301-430aa, and the upstream and downstream were designed. The primers are F1, R1; the FnBPAr10-11 (801-874aa) in the FnBPAr1-11 region is named as FnBPA801-874aa, and the upstream and downstream primers are designed as F2, R2; the artificial peptide rigid Linker (LAEAAAKEAAAKAAA) is designed to combine FnBPA801-874aa and FnBPA301 The -430aa fusion, named FL, was designed to connect F3 by the overlapPCR method, and the upstream and downstream primers of F10 were P2 and P3 respectively; the specific Primersofcoding, Primerlocation, Primersequence and the length of the amplified fragment are shown in Table ...

Embodiment 2

[0069] This example illustrates the preparation of S. aureus TAF fusion proteins.

[0070] 1. According to the FL obtained in this experiment and the protein HlaD and Trap obtained in the previous laboratory, on the premise that the gene sequence is correct, according to the published gene sequence, Primer5 software is used to design primers and artificial peptide Linker. For details, see Table 2 and the schematic diagram of the amplification method ( Figure 10 ) shown. In the table, the underline is the restriction site, and the primer is italicized as Linker. The primers PT and RT amplify the target protein Trap, the primers PH and RH amplify the target protein HlaD, and the primers PF and RF amplify the target protein FL.

[0071] Table 2 Design scheme of primers and linker

[0072]

[0073]

[0074] 2. Amplification of Trap, HlaD and FL target genes

[0075] PCR amplification method: put the micro-reaction tube at low temperature, add the reagents used according...

Embodiment 3

[0089] This example illustrates the comparison of the immune effects of TAF, Trap, HlaD and FL proteins.

[0090] 1. Animal immunization and challenge test

[0091] A total of 180 healthy, female, and same-week-old SPF ICR mice were divided into abdominal cavity group and lung group. The abdominal cavity group was divided into Trap experimental group, FL experimental group, TAF experimental group, mixed protein group (Mixture group) and Mock group, with 20 animals in each group; lung group was divided into HlaD experimental group, TAF experimental group, mixed protein group ( Mixture group) and Mock group, 20 in each group. Each experimental histone was purified at 1 mg / ml, emulsified with Freund's complete adjuvant in a ratio of 1:1, and immunized by intramuscular injection of 200 μl of each mouse leg; 21 days after the first immunization, the protein was mixed with incomplete Freund's adjuvant with Emulsify in a 1:1 ratio and boost immunity by intramuscular injection in th...

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Abstract

The invention relates to a staphylococcus aureus FnBPA immunodominance segment and provides a coding gene of the immunodominance segment. The invention also relates to a fusion protein containing the staphylococcus aureus FnBPA immunodominance segment. The fusion protein is prepared by fusing TRAP protein of staphylococcus aureus, alpha-hemolysin immunodominance segment, and staphylococcus aureus FnBPA immunodominance segment. The invention also provides a coding gene, a preparation method and applications of the fusion protein. The TAF fusion protein has been applied to mouse immunity and challenge tests, and the test results show that TAF fusion protein has a good immuno-protective effect, is an ideal vaccine antigen of staphylococcus aureus, and has an important value in the development and application of novel vaccines.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for preparing TAF fusion protein of Staphylococcus aureus and its application. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus, S.aureus) can cause a variety of human infections, and is also an important pathogen of cow mastitis. With the emergence of a large number of drug-resistant strains and the loss of efficacy of antibiotics, vaccination has become the best choice to prevent S.aureus infection. Staphylococcus aureus produces a large number of pathogenic factors, which can cause disease through factors such as adhesion, immune evasion, and production of invasive toxins and enzymes. Therefore, an effective S.aureus vaccine should be a multi-antigen vaccine that simultaneously blocks the above links . [0003] Staphylococcus aureus FnBPA is an important adhesion molecule and has good antigenicity. Trap has an anti-oxidative ...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K19/00C12N15/31C12N15/62A61K39/085A61P31/04
CPCA61K39/085A61K2039/54A61K2039/545C07K14/31C07K2319/00
Inventor 于立权崔玉东刘道龙
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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