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Recombined staphylococcus aureus enterotoxin Q oral preparation and application thereof

A technology of Staphylococcus aureus enterotoxin and Staphylococcus enterotoxin, applied in the field of bioengineering, can solve the problems of lack of strict quality control standards, simplicity, and easy introduction of other protein impurities

Inactive Publication Date: 2008-10-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned method for extracting enterotoxin from the fermentation metabolites of Staphylococcus aureus has certain deficiencies, such as simple preparation process, lack of strict quality control standards, instability between batches, and easy introduction of other protein impurities, etc., thereby affecting the purity of the final product , activity and quality

Method used

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  • Recombined staphylococcus aureus enterotoxin Q oral preparation and application thereof
  • Recombined staphylococcus aureus enterotoxin Q oral preparation and application thereof
  • Recombined staphylococcus aureus enterotoxin Q oral preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: PCR amplification of the gene sequence encoding the mature peptide of SEQ protein containing restriction sites:

[0049] Design the following primer sequences:

[0050] SEQ ID NO.3: at g gat cc g atg tag ggg taa tc (the underlined part is the restriction site of BamH I)

[0051] SEQ ID NO.4: gc c tcg ag t tat tca gtt ttc tca t (the underlined part is the Xho I restriction site)

[0052] The Staphylococcus aureus (FRI 1230) genome was used as a template to amplify the mature peptide gene fragment seq of Staphylococcus aureus enterotoxin SEQ.

[0053] The conditions for PCR amplification are as follows. The gene fragment encoding the mature peptide of SEQ is amplified, wherein each gene fragment has a BamH I restriction site at the 5' end and an Xho I restriction site after the terminator at the 3' end.

[0054] PCR system:

[0055] h 2 O: 60 μL

[0056] Buffer(10×): 10μL

[0057] Mg 2+ (25mmol / L): 8μL

[0058] BSA (5mg / mL): 10μL

[0059] Primer-...

Embodiment 2

[0070] Example 2: Construction of recombinant plasmid pGEM-T-SEQ encoding enterotoxin SEQ protein mature peptide gene seq containing restriction sites:

[0071]The gene fragment encoding the mature peptide of SEQ protein with restriction sites obtained by PCR amplification was cloned into the pGEM-T plasmid to construct the pGEM-T-SEQ recombinant plasmid, and the above recombinant plasmid was transformed into E. coli DH5α for amplification. Extract the above recombinant pGEM-T-SEQ plasmid, send it for sequencing after enzyme digestion and identification, and the sequencing results show that the gene sequence encoding the mature peptide of the SEQ protein with the enzyme digestion site obtained above is different from the corresponding gene sequence on GenBank except at the 5' The rest of the site is completely consistent except for the addition of a restriction site, and the corresponding amino acid sequence of the enterotoxin Q mature peptide has only two more amino acids (Gly...

Embodiment 3

[0072] Example 3: Construction of the pGEX-4T-1-SEQ recombinant expression plasmid containing the gene encoding the mature peptide of SEQ with a restriction site:

[0073] The pGEM-T-SEQ recombinant plasmid and the pGEX-4T-1 plasmid obtained in Example 2 containing the gene fragment encoding the mature peptide of the SEQ protein obtained in Example 2 were respectively digested with BamH I and Xho I. The gene fragment encoding the mature peptide of SEQ containing the restriction site and the large fragment of the digested product of the pGEX-4T-1 plasmid after digestion were respectively recovered and ligated to construct the expression plasmid pGEX-4T-1-SEQ. The above-mentioned recombinant expression plasmid was transferred into Escherichia coli DH5α to amplify and extract the plasmid, and after BamH I and Xho I digestion and identification, the result showed that the target gene fragment had been inserted into the vector plasmid, and the electrophoresis results were shown in F...

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Abstract

The invention provides a recombinant staphylococcal enterotoxin Q oral preparation, the recombinant staphylococcal enterotoxin Q has SEQ ID NO.1 amino acid sequence, and the oral preparation further comprises a pharmaceutical allowable drug excipient or a carrier. The oral preparation proves that the protein can enter the systemic blood circulation by penetrating epithelial cells on small intestine with the form of complete molecules and maintain the super-antigen activity for promoting the spleen lymphocyte proliferation and inhibiting the growth of tumor cells, as well as the application in the preparation of drugs for treating malignant tumors and other serious complications by the Caco-2 monolayer cell transmembrane transport test.

Description

technical field [0001] The invention belongs to bioengineering, and relates to the preparation of recombinant staphylococcal enterotoxin Q (Staphylococcal Enterotoxin Q, SEQ), the preparation of oral preparation with the main active ingredient of SEQ, and its application in tumor treatment. Specifically, the method of genetic engineering is used to prepare high-purity recombinant Staphylococcus aureus enterotoxin Q protein and related protein products, and prepare them into oral preparations for the treatment and rehabilitation of tumor patients. Background technique [0002] Staphylococcal Enterotoxin is produced by Staphylococcus aureus. More than 20 types of enterotoxins have been identified, including classic enterotoxins such as SEA, SEB, SEC, SED, SEE, etc., and newly discovered enterotoxins SEG-SEQ. The molecular weight of different types of enterotoxins ranges from 25 to 33 kD, and the amino acid sequences of various types of enterotoxins have certain homology (28% ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/085C12N15/31C12N15/70C07K14/31A61P35/00A61P37/04
Inventor 陈枢青丁丁
Owner ZHEJIANG UNIV
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