Antigenically-changed enterotoxin C2 mutant, coding gene thereof, preparation thereof and application thereof

A technology encoding genes and enterotoxins, applied in the field of genetic engineering, can solve the problems affecting clinical treatment effect, T lymphocyte reduction, etc., to avoid neutralization and seroclearance effect, high tumor inhibitory activity, and good superantigen activity. Effect

Inactive Publication Date: 2012-08-15
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a heterologous toxin protein, wild-type SEC2 stimulates the body's immune system and induces B cells to produce a large number of antibodies. These antibodies can specifically recognize and bind to the antigenic epitope of wild-type SEC2, making it recognized by serum Antibody neutralization, which leads to a greatly reduced ability to stimulate and activate T lymphocytes in vivo, thus affecting its clinical therapeutic effect

Method used

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  • Antigenically-changed enterotoxin C2 mutant, coding gene thereof, preparation thereof and application thereof
  • Antigenically-changed enterotoxin C2 mutant, coding gene thereof, preparation thereof and application thereof
  • Antigenically-changed enterotoxin C2 mutant, coding gene thereof, preparation thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The coding gene sequence of an enterotoxin C2 mutant having the sequence listing SEQ ID NO: 1 base sequence (see the sequence listing):

[0029] 001GAGAGTCAAC CAGACCCTAC GCCAGATGAGTTGCACAAAT

[0030] 041CAAGTGAGTT TACTGGTACG ATGGGTAATA TGAAAGTATT

[0031] 081ATATGATGAT CATTATGTAT CAGCAACTAA AGTTATGTCT

[0032] 121 GTAGATAAAT TTTTGGCACA TGATTTAATT TAIAACATTA

[0033] 161 GTGATAAAAA ACTAAAAAAT TATGACAAAG TGAAAACAGA

[0034] 201GTTATTAAATGAAGATTTAGCAAAGAAGTACAAAGATGAA

[0035] 241 GTAGTTGATG TGTATGGATC AAATTACTAT GTAAACTGCT

[0036] 281ATTTTTCATC CAAAGATAAT GTAGGTAAAGTTACAGGTGG

[0037] 321 TAAAACTTGT ATGTATGGAG GAATAACAAA ACATGAAGGA

[0038] 361AACCACTTTG ATAATGGGAA CTTACAAAAT GTACTTAIAA

[0039] 401GAGTTTATGA AAATAAAAAGA AACACAATTT CTTTTGAAGT

[0040] 441GCAAACTGAT AAGAAAAGTG TAACAGCTCA AGAACTAGAC

[0041] 481ATAAAAGCTA GGAATTTTTT AATTAATAAA AAAAATTTGT

[0042] 521ATGAGTTTAA CAGTTCACCATATGAAACAG GATATATAAA

[0043] 561ATTTATTGAA AATAACGGCA ATACTTTTTG GTATGATAT...

Embodiment 2

[0073] A preparation method of enterotoxin C2 mutant with changed antigenic epitope:

[0074] ① Extraction of Staphylococcus aureus genomic DNA

[0075] Inoculate a single colony of Staphylococcus aureus in 5ml of liquid LB medium, culture overnight on a shaker at 37°C, and collect 1.5ml of the culture by centrifugation to collect the bacteria. Extract Staphylococcus aureus genomic DNA (genome DNA extraction operation according to F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G Seidman, J.A. Smith, K. Stellar "fine Compilation of Molecular Biology Experiment Guide, New York John Wiley & Sons Publishing House, 1995, third edition, P39-40).

[0076] ②PCR primer design and reaction conditions:

[0077] The sequences of PCR primers were designed and synthesized as follows:

[0078] sec2-F: 5'-CGGAATTCGAGAGTCAACCAGA-3'

[0079] sec2-R: 5'-TCGCTCGAGTTATCCATCTTTGTTG-3'

[0080] F-Mutant: 5'-GGGTAATACGAAAGTATTATATGATGATC-3'

[0081] R-Mutant: 5'-CATCATATAATACTTTCGTATTACCCATCG...

Embodiment 3

[0123] Detection of Superantigen Activity of Enterotoxin C2 Mutant with Changed Antigen Epitope

[0124] The SPF-grade pure-line mouse Balb / c was sacrificed through the cervical spine, and the spleen was collected under aseptic conditions, crushed lightly, and passed through a 200-mesh sieve. Then the cell suspension passed through the sieve was centrifuged at 1000rpm / min for 5min to collect the cell pellet, the cells were resuspended with 5mL red blood cell lysate, left to stand for 5min and then centrifuged at 1000rpm / min for 5min. Then wash the cells 1-2 times with serum-free 1640 medium (purchased from Gibco), and finally adjust the cell concentration with RPMI-1640 medium containing 10% (volume percentage) calf serum (purchased from Gibco). Take 8×10 5 cells / well added to 96-well plate. The purified mutants were added to each well at a final concentration of 10ng / ml, 100ng / ml, 1000ng / ml, and 10000ng / ml. Three replicate wells were set up for each concentration, and SEC2 ...

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Abstract

An antigenically-changed enterotoxin C2 mutant, a coding gene thereof, preparation thereof and application thereof relate to the technical field of gene engineering, in particular to an antigenically-changed enterotoxin C2 mutant with officinal prospect, a coding gene thereof, preparation thereof and application thereof. An amino acid sequence of the antigenically-changed enterotoxin C2 mutant is shown in a sequence listing SEQ ID NO: 3, and a base sequence of the coding gene of the enterotoxin C2 mutant is shown in a sequence listing SEQ ID NO: 1.

Description

Technical field: [0001] The present invention relates to the technical field of genetic engineering, and more specifically relates to an antigenically altered enterotoxin C2 mutant with medicinal prospects, its coding gene, its preparation and application. Background technique: [0002] Superantigen (SAg) is a group of protein molecules encoded by bacteria or viruses, which have strong immunostimulatory activity on T lymphocytes at very low concentrations. Unlike traditional antigens, superantigens do not require the presentation of antigen-presenting cells, and the antigen-binding region on the outside of antigen-presenting cells combines with MHC II (histocompatibility complex) molecules and T cell Vβ regions to form a complex. Thereby a large number of T lymphocytes are stimulated to proliferate, which in turn leads to the release of a large number of cytokines and other effector molecules in vitro or in vivo. Because of the special biological activity and mechanism of a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70A61K38/16A61P35/00A61P37/02
Inventor 徐明恺王洪波谭焕波张惠文李旭张成刚
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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