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Recombinant Staphylococcus aureus enterotoxin J oral preparation and use

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: 2008-12-17
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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  • Recombinant Staphylococcus aureus enterotoxin J oral preparation and use
  • Recombinant Staphylococcus aureus enterotoxin J oral preparation and use
  • Recombinant Staphylococcus aureus enterotoxin J oral preparation and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: PCR amplification of the gene sequence encoding SEJ protein mature peptide containing restriction sites:

[0048] Design the following primer sequences:

[0049] SEQ ID NO.3: gca gat agg aaa aat g (the underlined part is the restriction site of BamH I)

[0050] SEQ ID NO.4: cgc cta cag aac caa a (the underlined part is the Xho I restriction site)

[0051] The genome of Staphylococcus aureus (FRI 1230) was used as a template to amplify the mature peptide gene fragment sej of Staphylococcus aureus enterotoxin SEJ.

[0052] The conditions for PCR amplification were as follows. The gene fragments encoding SEJ 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.

[0053] PCR system:

[0054] h 2 O: 60 μL

[0055] Buffer(10×): 10μL

[0056] Mg 2+ (25mmol / L): 8μL

[0057] BSA (5mg / mL): 10μL

[0058] Primer-up (25μmol / L): 4μL

[0059] Prim...

Embodiment 2

[0069] Example 2: Construction of recombinant plasmid pGEM-T-SEJ encoding enterotoxin SEJ protein mature peptide gene sej containing restriction sites:

[0070]The gene fragment encoding the mature peptide of SEJ protein with restriction sites obtained by PCR amplification was cloned into the pGEM-T plasmid to construct the pGEM-T-SEJ recombinant plasmid, and the above recombinant plasmid was transformed into E. coli DH5α for amplification. The above recombinant pGEM-T-SEJ 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 SEJ 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 exactly the same except for the addition of a restriction site, and the corresponding amino acid sequence of the enterotoxin J mature peptide has only two more amino acids (Gly-Ser...

Embodiment 3

[0071] Example 3: Construction of pGEX-4T-1-SEJ recombinant expression plasmid containing a gene encoding SEJ mature peptide with a restriction site:

[0072] The pGEM-T-SEJ recombinant plasmid and the pGEX-4T-1 plasmid obtained in Example 2 containing the gene fragment encoding the mature peptide of the SEJ protein obtained in Example 2 were respectively digested with BamH I and Xho I. The gene fragment encoding the mature peptide of SEJ containing the restriction site and the large fragment of the digested product of pGEX-4T-1 plasmid after digestion were recovered and ligated to construct the expression plasmid pGEX-4T-1-SEJ. The above recombinant expression plasmid was transformed into Escherichia coli DH5α 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 results of electrophoresis are shown in the attached image 3 , lane 1 in the figur...

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Abstract

The invention provides a recombinant staphylococcus aureus enterotoxin J oral preparation which has an amino acid sequence of SEQ ID NO.1 and also comprises excipients in medicine or carriers allowed by the preparation. By the experiment on the Caco-2 monolayer cell trans-membrane transport, the preparation of the invention proves that the proteins can enter the general blood circulation from intestinal epithelial cells in the form of complete molecules and keep promoting the splenic lymphocyte proliferation and preventing the superantigen activity for the growth of tumor cells, and can be applied to the preparation of drugs for curing the malignant tumor and other serious complications.

Description

technical field [0001] The invention belongs to bioengineering, and relates to the preparation of recombinant staphylococcal enterotoxin J (Staphylococcal Enterotoxin J, SEJ), the preparation of oral preparation with SEJ as the main active ingredient and its application in tumor treatment. Specifically, it uses genetic engineering to prepare high-purity recombinant Staphylococcus aureus enterotoxin J protein and related protein products, and prepares 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%). Accor...

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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