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A kind of artificial protein with characteristics of Vibrio cholerae toxin a subunit and Staphylococcus aureus and its application

A technology of vibrio cholerae toxin and staphylococcus, which is applied in the field of artificial protein, can solve the problems of strong toxicity and achieve the effect of strong specificity, good immunogenicity and significant market value

Active Publication Date: 2020-01-17
BEIJING DIRECTION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are two problems in administering soluble antigens via the mucosal route: how to maintain the integrity of the antigen in the rough mucosal environment, and how to enhance the immunogenicity of the antigen
However, because CT and LT are too toxic, they cannot be used directly on humans.

Method used

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  • A kind of artificial protein with characteristics of Vibrio cholerae toxin a subunit and Staphylococcus aureus and its application
  • A kind of artificial protein with characteristics of Vibrio cholerae toxin a subunit and Staphylococcus aureus and its application
  • A kind of artificial protein with characteristics of Vibrio cholerae toxin a subunit and Staphylococcus aureus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation and purification of artificial protein protein with Vibrio cholerae toxin A subunit and Staphylococcus aureus characteristics

[0027] The DNA sequence (shown in SEQ ID NO.2) corresponding to the protein sequence confirmed by DNA analysis and protein structure analysis was synthesized, and Nde I and Xho I restriction sites were introduced, and the commercial pET30a plasmid was used as the basic vector to construct pCTA, the protein expressed by this expression vector has a HIS tag at the C-terminus of the protein, which is conducive to the later purification of affinity chromatography.

[0028] (1) Preparation of Escherichia coli BL21DE3 Competent Cells

[0029] 1. Pick a single colony from a fresh plate cultivated overnight at 37°C, inoculate it in 3ml of LB liquid medium without antibiotics, and cultivate it with shaking at 240rpm at 37°C for 4-5 hours.

[0030] 2. Take 0.8ml of the above-mentioned culture and add it to 200ml LB medium without a...

Embodiment 2

[0080] Example 2 Cell Membrane Targeting of Artificial Protein Protein with Vibrio cholerae Toxin A Subunit and Staphylococcus Aureus Characteristics

[0081] Ganglioside-binding activity analysis of an artificial protein with toxin A subunit of Vibrio cholerae and Staphylococcus aureus demonstrating good cell membrane targeting sex.

[0082] Coat a polyethylene plastic plate with a concentration of 3ug / mL ganglioside, and at the same time use distilled water, other fusion proteins and BSA as controls, repeat 3 wells respectively, discard the coating after coating overnight at 4°C, and use blocking solution (dissolving Block with 1% BSA in PBS solution) at 37°C for 1 h, discard the coating, wash with PBS for 3 times, add purified recombinant CTA (about 50 ng), and use 1% BSA as a negative control, incubate at 37°C for 1 h, wash with PBS 3 times, add rabbit anti-CT serum (1:4000 dissolved in blocking solution), incubate at 37°C for 1h, wash with PBS 3 times, each time blot dry...

Embodiment 3

[0083] Example 3 The immune effect of the artificial protein with the characteristics of Vibrio cholerae toxin A subunit and Staphylococcus aureus

[0084] The animal nasal drop immunization scheme of the artificial protein with the characteristics of Vibrio cholerae toxin A subunit and Staphylococcus aureus and Ebola surface glycoprotein (GP), verified that with the assistance of the protein of the present invention, it can be immunized by nasal drop Produce high titers of neutralizing antibodies specific to GP antigens.

[0085] Antigen adjuvant compatibility and immunization dose and procedure are as follows:

[0086] 1 Experimental materials Stone crab monkeys: 4 females and 2 males, a total of 6

[0087] Protein of the present invention (purity higher than 95%)

[0088] GP antigen (purity higher than 95%)

[0089] 2 Immunization steps

[0090] The stone crab monkeys were randomly divided into groups to ensure that there were 2 females and 1 male in each group, numbere...

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Abstract

The invention provides artificial protein having vibrio cholerae toxin A-subunit and staphylococcus aureus characteristics and an application thereof, which belong to the technical field of protein engineering. According to the invention, the artificially designed vibrio cholerae toxin A-subunit and staphylococcus aureus DNA sequences are subjected to synthesis, an amino acid sequence is shown as SEQ ID NO.1, and a nucleotide sequence is shown as SEQ ID NO.2. The fusion protein has the advantages of good immunogenicity and strong specificity, can effectively introduce into an biological body through a mucous membrane, and a neutralizing antibody is generated in the body, the neutralizing antibody can be used for preparing a biological product as an immunologic adjuvant, and the market value is obvious.

Description

technical field [0001] The invention relates to the field of virus immunity, in particular to an artificial protein with the characteristics of Vibrio cholerae toxin A subunit and Staphylococcus aureus and its application. Background technique [0002] Immune adjuvants are also called non-specific immune proliferators. It is not antigenic in itself, but it can enhance immunogenicity (see antigen) or change the type of immune response together with antigen or pre-injection into the body. [0003] There are many kinds of immune adjuvants, and there is no unified classification method at present. Commonly used adjuvants can be divided into 4 categories: inorganic adjuvants, such as aluminum hydroxide, alum, etc.; organic adjuvants, microorganisms and their products such as mycobacteria (tuberculosis Bacillus, BCG), Brevibacterium, Bacillus pertussis, endotoxin, bacterial extract (muramoyl dipeptide), etc.; synthetic adjuvants, such as artificially synthesized double-stranded p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/39
CPCA61K39/12A61K39/39A61K2039/543A61K2039/55544C07K14/28C07K14/31C07K2319/00C12N2760/14134
Inventor 曹健荣
Owner BEIJING DIRECTION BIOTECH CO LTD
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