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Recombined staphylococcus aureus enterotoxin K 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 simplicity, instability between batches, 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 K oral preparation and application thereof
  • Recombined staphylococcus aureus enterotoxin K oral preparation and application thereof
  • Recombined staphylococcus aureus enterotoxin K 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 SEK protein containing restriction sites:

[0047] Design the following primer sequences:

[0048] SEQ ID NO.3: gga tcc caa ggt gat ata gga a (the underlined part is the restriction site of BamH I)

[0049] SEQ ID NO.4: ctc gag taa tag gtt tat ttt gtt (the underlined part is the Xho I restriction site)

[0050] The genome of Staphylococcus aureus (FRI 315) was used as a template to amplify the mature peptide gene fragment sek of Staphylococcus aureus enterotoxin SEK.

[0051] The conditions for PCR amplification were as follows. The gene fragments encoding SEK mature peptides were amplified, wherein each gene fragment had 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 (25μmol...

Embodiment 2

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

[0069] The gene fragment encoding the mature peptide of SEK protein with restriction sites obtained by PCR amplification was cloned into the pGEM-T plasmid to construct the pGEM-T-SEK recombinant plasmid, which was transformed into Escherichia coli DH5α for amplification. The above recombinant pGEM-T-SEK 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 SEK protein with the restriction site obtained above was different from the corresponding gene sequence on GenBank except at the 5' The amino acid sequence of the corresponding enterotoxin K mature peptide has two more amino acids (Gly -Ser), at the same time, the following amino acids were substituted: ILe 21 -Val 21 ;Asn 22 -Asp 22 ;Ile 84 -Val 84 ...

Embodiment 3

[0070] Example 3: Construction of pGEX-4T-1-SEK recombinant expression plasmid containing a gene encoding SEK mature peptide with a restriction site:

[0071] The pGEM-T-SEK recombinant plasmid and pGEX-4T-1 plasmid obtained in Example 2 containing the gene fragment encoding the mature peptide of the SEK protein obtained in Example 2 were respectively digested with BamH I and Xho I. The gene fragment encoding the mature peptide of SEK 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-SEK. The above-mentioned recombinant expression plasmid was transferred into Escherichia coli DH5α to amplify and extract the plasmid. After digestion and identification by BamH I and Xho I, the results 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 K oral preparation, the recombinant staphylococcal enterotoxin K 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 K (Staphylococcal Enterotoxin K, SEK), the preparation of oral preparation with SEK as the main active ingredient and its application in tumor treatment. Specifically, it is to use genetic engineering to prepare high-purity recombinant Staphylococcus aureus enterotoxin K 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%). Ac...

Claims

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Application Information

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