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Polypeptide WA8 for inhibiting type-2 shiga toxin activity and coding gene and application thereof

A Shiga toxin, coding gene technology, applied to peptides, peptide/protein components, recombinant DNA technology, etc.

Active Publication Date: 2011-01-19
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Peptide inhibitors against Shiga toxin are rarely reported

Method used

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  • Polypeptide WA8 for inhibiting type-2 shiga toxin activity and coding gene and application thereof
  • Polypeptide WA8 for inhibiting type-2 shiga toxin activity and coding gene and application thereof
  • Polypeptide WA8 for inhibiting type-2 shiga toxin activity and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Modification, chemical synthesis and binding activity analysis of short peptides TF1 (also known as P1) and WA8 (also known as P8)

[0027] 1. Modification and synthesis of short peptides TF1 (also known as P1) and WA8 (also known as P8)

[0028] 1. Screening of TF1 and WA8 synthetic peptide sequences

[0029] Preparation of Stx2B protein: using EHEC O157:H7 bacteria (purchased from ATCC) as a template, the stx2B gene was amplified with primers 5'-catatggctaaaggtaaaattgag-3' and 5'gcggccgcgcctcagtcatcagtcatt-3'. The pEASY-T1 clone vector was used for sequencing identification, and the sequencing results showed that the nucleotide sequence of the amplified product was as the 1353327th to 1353533rd positions of GenbankNC_002655. The amplified product was inserted into the Nde I and Not I restriction sites of the pET22b+ vector (purchased from Novagen) to form the recombinant plasmid pET22b-stx2B, and then pET22b-stx2B was introduced into the E.coli.BL21 (DE3) st...

Embodiment 2

[0043] Example 2. In vitro cytotoxicity inhibitory activity of TF1 and WA8 synthetic peptides and binding blocking effect on target cells of toxins

[0044] HeLa cell line, trypsin digestion solution and double antibody were purchased from Hyclone Company, DMEM medium was purchased from GIBCO Company, fetal bovine serum was purchased from Biochorm Company, MTT was from Sigma Company, and FITC was purchased from Leybold Company. Cell culture flasks and culture plates were purchased from CORNING Company, CO 2The incubator is a product of SANYO Company of Japan, the inverted optical microscope is a product of Olympus Company of Japan, and the flow cytometer is a product of Coulter Company.

[0045] 1. Cytotoxicity neutralization experiment

[0046] Collect the cultured logarithmic phase cells (HeLa cells), adjust the concentration of the cell suspension, add 100 μl to each well, and plate to adjust the density of the cells to be tested to 10 4 / well (edge ​​wells filled with st...

Embodiment 3

[0049] Example 3. Protective effect of TF1, WA8 synthetic peptides on type 2 Shiga toxin-challenged mice

[0050] Male Balb / C (17-19g) mice were provided by the Animal Center of Academy of Military Medical Sciences. Test polypeptide TF1 (also known as P1) and WA8 (also known as P8) purity> 95% (g grade)

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PUM

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Abstract

The invention discloses a polypeptide WA8 for inhibiting type-2 shiga toxin activity and a coding gene and application thereof. The polypeptide provided by the invention is named as WA8 (also named P8), and the amino acid sequence of the polypeptide is expressed as a sequence 2 in a sequence table. The polypeptide P8 can be prepared into a medicament for preventing and / or treating diseases caused by type-2 shiga toxin or pathogenic bacteria which can generate the type-2 shiga toxin.

Description

technical field [0001] The present invention relates to polypeptide WA8 for inhibiting the activity of type 2 Shiga toxin, its coding gene and application. Background technique [0002] Shiga toxin (Stx), also known as Shiga-like toxin (Slt), is a kind of bacterial exotoxin with enterotoxicity, cytotoxicity and neurotoxicity produced by enteropathogenic bacteria, including type 1, Type 2 Shiga toxins (Stx1, Stx2) are the most critical potent virulence factors for emerging enteric pathogens such as acute bacillary dysentery, cholera O139, O157:H7, and can cause hemorrhagic colitis (HC ), thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) with a high mortality rate and other serious complications. Both epidemiological studies and clinical data have shown that compared with Stx1, Stx2 has a higher correlation with infection complications, especially the occurrence of HUS, and is considered to be an important target for prevention and treatment drug d...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K38/10A61P31/04
CPCY02A50/30
Inventor 王慧李涛侯晓军王琴屠伟刘越男史晶蔡昆
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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