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Recombined staphylococcus aureus enterotoxin N oral preparation and application thereof

A technology of Staphylococcus aureus enterotoxin and Staphylococcus enterotoxin, applied in the field of bioengineering, can solve problems affecting product purity, activity and quality, instability between batches, and simplicity

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: PCR amplification of the gene sequence encoding the mature peptide of SEN protein containing restriction sites:

[0047] Design the following primer sequences:

[0048] SEQ ID NO.3: gga tcc gaa gta gac aaa aaa ga (the underlined part is the restriction site of BamH I)

[0049] SEQ ID NO.4: ctc gag ata atc ​​atc aat cac tta (the underlined part is the Xho I restriction site)

[0050] The genome of Staphylococcus aureus (FRI 1230) was used as a template to amplify the mature peptide gene fragment sen of Staphylococcus aureus enterotoxin SEN;

[0051] The PCR amplification conditions are as follows, the gene fragments encoding the mature peptide of SEN are 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.

[0052] PCR system:

[0053] h 2 O: 60 μL

[0054] Buffer(10×): 10μL

[0055] Mg 2+ (25mmol / L): 8μL

[0056] BSA (5mg / mL): 10μL

[0057] Primer-up ...

Embodiment 2

[0068] Example 2: Construction of the recombinant plasmid pGEM-T-SEN encoding enterotoxin SEN protein mature peptide gene sen containing restriction sites:

[0069]The gene fragment encoding the mature peptide of SEN protein with restriction sites obtained by PCR amplification was cloned into the pGEM-T plasmid to construct the pGEM-T-SEN recombinant plasmid, and the above recombinant plasmid was transformed into E. coli DH5α for amplification. The above recombinant pGEM-T-SEN plasmid was extracted, identified by enzyme digestion, and then sent for sequencing. The sequencing results showed that the gene sequence encoding the mature peptide of the SEN protein with the enzyme digestion site obtained above was 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. The amino acid sequence of the corresponding enterotoxin N mature peptide has only two more amino acids (Gl...

Embodiment 3

[0070] Example 3: Construction of pGEX-4T-1-SEN recombinant expression plasmid containing the gene encoding SEN mature peptide with restriction sites:

[0071] The pGEM-T-SEN recombinant plasmid and pGEX-4T-1 plasmid obtained in Example 2 containing the gene fragment encoding the mature peptide of the SEN protein obtained in Example 2 were respectively digested with BamH I and Xho I. The gene fragment encoding the mature peptide of SEN containing the restriction site and the large fragment of the digested product of the pGEX-4T-1 plasmid after digestion were recovered and ligated to construct the expression plasmid pGEX-4T-1-SEN. The above-mentioned recombinant expression plasmid was transferred into Escherichia coli DH5a to amplify and extract the plasmid. After digestion and identification by BamH I and Xho I, the result showed that the target gene fragment had been inserted into the vector plasmid. The electrophoresis results are shown in Figure 3, lane 1 in the figure: nucl...

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Abstract

The invention provides a recombinant staphylococcal enterotoxin N oral preparation, the recombinant staphylococcal enterotoxin N 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 N (Staphylococcal Enterotoxin N, SEN), the preparation of oral preparation with the main active ingredient of SEN and its application in tumor treatment. Specifically, it is to use genetic engineering to prepare high-purity recombinant Staphylococcus aureus enterotoxin N 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% to 98%). A...

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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