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Two staphylococcus aureus protein antigens and preparation method and use thereof

A Staphylococcus, aureus technology, applied in the fields of molecular biology and immunology, can solve the problems of small expression and difficult purification steps

Active Publication Date: 2016-03-02
CHANGCHUN BCHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] However, due to the excessive molecular weight of IsdB natural protein, its expression in the E. coli genetic engineering expression system is small, and the subsequent purification steps are more difficult.

Method used

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  • Two staphylococcus aureus protein antigens and preparation method and use thereof
  • Two staphylococcus aureus protein antigens and preparation method and use thereof
  • Two staphylococcus aureus protein antigens and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Synthesis of Nucleotide Sequence Encoding Staphylococcus aureus alpha-hemolysin Attenuated Protein and Obtaining of Clones Containing the Same

[0054] The amino acid sequence of natural Staphylococcus aureus α-hemolysin (GenBank accession number: AAA26598.1) was obtained through network search, as shown in SEQ ID NO: 1. The sequence was modified as follows: histidine (His, H) at position 35 was replaced by leucine (Leu, L) to obtain the S. aureus alpha-hemolysin mutation shown in SEQ ID NO: 3 The amino acid sequence of the body (HlaM35).

[0055] The nucleotide sequence encoding the amino acid sequence of HlaM35 was optimized by selecting the preferred codons of Escherichia coli, and the nucleotide sequence of SEQ ID NO: 4 was obtained by chemical synthesis. NdeI was added upstream of the nucleotide sequence of SEQ ID NO: 4, and an XhoI restriction site was added downstream. Thereby a nucleotide sequence for protein expression is obtained.

[0056] The ob...

Embodiment 2

[0057] Example 2. Obtainment of the nucleotide sequence encoding native S. aureus alpha-hemolysin (HlaWT) and obtaining of clones containing the same

[0058] The primers were designed, and the nucleotide sequence shown in SEQ ID NO: 4 was used as a template for mutation by the method of PCR to obtain the nucleotide sequence encoding HlaWT shown in SEQ ID NO: 2.

[0059] Mutation primers are as follows:

[0060] HlaWT-1:

[0061]5’-CGATAAAGAAAACGGTATGCACAAGAAAGTTTTTCTACTCTTTCA-3’

[0062] HlaWT-2:

[0063] 5’-TGAAAGAGTAGAAAACTTTCTTGTGCATACCGTTTTCTTTATCG-3’

[0064] PCR system: 1 μl (10 ng) of the PET20b-HlaM35 plasmid template DNA obtained above, 2 μl each of mutant primers HlaWT-1 and HlaWT-2 (0.5 μM concentration), 1 μl of dNTP, 0.5 μl of PfuTurbo DNA polymerase (2.5 U / μl, purchased from Takera), 5 μl of 10× reaction buffer, 38.5 μl of deionized water, and a total volume of 50 μl.

[0065] Mutation PCR conditions: preheating at 95°C for 5 min; 95°C, 1 min, 58°C, 1 min, ...

Embodiment 3

[0067] Example 3. Synthesis of Nucleotide Sequences Encoding Full Length IsdB Protein and Obtaining of Clones Containing the Same

[0068] The amino acid sequence of Staphylococcus aureus iron-regulated surface determinant protein B (IsdB) was obtained through network retrieval, as shown in SEQ ID NO: 5.

[0069] The preferred codons of Escherichia coli were selected to optimize the nucleotide sequence encoding the amino acid sequence of IsdB, and the nucleotide sequence encoding IsdB of SEQ ID NO: 6 was obtained by chemical synthesis. NdeI was added upstream of the nucleotide sequence of SEQ ID NO: 6, and an XhoI restriction site was added downstream. Thereby a nucleotide sequence that can be used for protein expression is obtained.

[0070] The obtained nucleotide sequence identified by sequencing was double digested with NdeI and XhoI, and then ligated with the expression vector PET20b (purchased from Novagen) that had been digested with the same double enzyme, and then li...

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Abstract

The invention belongs to the field of molecular biology and immunology. The invention relates to three truncated proteins of a staphylococcus aureus iron regulation surface determining protein B (IsdB) and a staphylococcus aureus alpha-hemolysin (Hla) attenuated protein, and their coding genes, preparation method and use. The invention also relates to the combined use of the three truncated proteins and the alpha-hemolysin attenuated body.

Description

technical field [0001] The present invention belongs to the fields of molecular biology and immunology. In particular, the present invention relates to three truncated proteins of Staphylococcus aureus iron-regulated surface determinant B (IsdB) and a Staphylococcus aureus alpha-hemolysin (Hla) attenuated protein, and Its encoding gene, preparation method and application. The present invention also relates to the combined application of the three truncated proteins and an α-hemolysin attenuator. Background technique [0002] Staphylococcus aureus is a gram-positive coccus, belonging to the family Bacteriaceae and the genus Staphylococcus. A typical Staphylococcus aureus is spherical, with a diameter of about 0.8 μm, and is arranged in a bunch of grapes under the microscope. Staphylococcus aureus has no spores and flagella, and most have no capsule. [0003] Staphylococcus aureus is widely distributed in nature and can be found in the air, water, dust and human and animal...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N15/31C12N15/70C12N1/21A61K39/085A61P31/04
CPCA61K39/015A61K2039/54C07K14/31
Inventor 孔维吴永革孙天旭陈莹侯宏嘉
Owner CHANGCHUN BCHT BIOTECH
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