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Recombination BCG viable bacterium strain capable of expressing and secreting staphylococcus aureus enterotoxin protein, viable bacterium vaccine and construction method and application thereof

A technology of Staphylococcus aureus enterotoxin and live bacteria, applied in the biological field, can solve problems such as limited effects of radiotherapy and chemotherapy, difficulty in curing cancer patients, and easy metastasis of tumors

Active Publication Date: 2014-01-08
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the continuous improvement of these 3 treatments, many cancer patients are still difficult to be cured
Because the tumor itself is prone to metastasis and recurrence, the effect of traditional surgery, radiotherapy and chemotherapy is limited

Method used

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  • Recombination BCG viable bacterium strain capable of expressing and secreting staphylococcus aureus enterotoxin protein, viable bacterium vaccine and construction method and application thereof
  • Recombination BCG viable bacterium strain capable of expressing and secreting staphylococcus aureus enterotoxin protein, viable bacterium vaccine and construction method and application thereof
  • Recombination BCG viable bacterium strain capable of expressing and secreting staphylococcus aureus enterotoxin protein, viable bacterium vaccine and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Detoxification mutation of wild-type S. aureus enterotoxin SEA and SEC2 genes

[0035] According to relevant literature reports (Vladimir S, et al. Toxicon. 2004, 43: 433-438. etc.) and the prediction results of the epitope analysis website (http: / / www.epipredict.de / ), wild-type Staphylococcus aureus Enterotoxin SEA and SEC2 gene sequences (GenBank accession number: NM_001126112) were genetically modified, and the modification sites were the amino acid sites of SEA (D227A) and SEC2 (T20L; T20L / G22E; G22E / N23A; T20L / G22E / N23A) .

[0036] 1. Design Primers

[0037] Using Gene Tool software to design relevant mutation primers, the results are as follows:

[0038] (1) SEA gene D227A single site mutation primer

[0039] Forward:

[0040] 5'-tgctatatatttatatacaagttaagtcgacaagctt-3'

[0041] Reverse:

[0042] 5'-atatgcatgttttcagagttaatcgtttt-3'

[0043] (2) SEC2 gene T20L single site mutation primer

[0044] Forward:

[0045] 5'-tttaatgggtaatatgaaatatttatatgatgatca-3'...

Embodiment 2

[0089] Construction of BCG Shuttle Expression Vector

[0090] Take the mutant Staphylococcus aureus enterotoxin CTG gene as an example:

[0091] 1. Construction of pMN-α-CTG shuttle expression vector

[0092] Plasmid pMN234 is an E. coli-Mycobacterium shuttle expression vector, also containing the HSP60 promoter.

[0093] Introduction of alpha secretion signal peptide:

[0094]The pMN234 plasmid does not contain an alpha secretion signal peptide sequence. In order to allow the target protein to be secreted and expressed in mycobacteria, we inserted a mycobacterial alpha secretion signal peptide sequence downstream of the promoter of the pMN234 vector, with the sequence shown in SEQ ID NO: 11 Nucleotide sequence.

[0095] (1) According to the multiple cloning site requirements of the pMN234 shuttle expression vector, the α gene cloning primers were designed and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis.

[0096] Forward: 5'-CGC GGATCC ATGACAGACGTGAG...

Embodiment 3

[0128] Genetic transformation of BCG shuttle expression vector

[0129] BCG was cultured in M7H9+ADC medium to logarithmic growth phase. After pre-cooling on ice, BCG cells were collected by centrifugation at 6000 rmp for 5 min, and the cells were washed three times with 10% pre-cooled sterilized glycerol. Suspended with 10% glycerol to prepare BCG electroporation competent cells.

[0130] Extract the recombinant plasmid of pMN-α-CTG shuttle secretion expression vector, and adjust the concentration to 10μg / ml. Take 100μl of BCG competent cells, add them to a 0.2cm electric shock transformation cup, add 5μl of the shuttle expression secretion vector plasmid pSL-CTG or pMN-α-CTG at a concentration of 10μg / ml, mix gently and place on ice 10min. Electroporation was performed using an electroporator (eppendorf). Set the parameter voltage to 2.5KV, time to 5ms, and two electric shocks. Then quickly add antibiotic-free M7H9+ADC medium for 20 hours, collect BCG cells by centrifuga...

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Abstract

The invention provides a recombination BCG viable bacterium strain capable of expressing and secreting staphylococcus aureus enterotoxin protein, a viable bacterium vaccine and a construction method and application thereof, and relates to the technical field of biology, in particular to the technical field of genetic engineering. The novel viable bacterium vaccine capable of preventing and treating mankind tumor diseases is provided. The recombination BCG viable bacterium strain is provided first, and the recombination BCG viable bacterium strain is a recombination strain which is obtained after a wild type staphylococcus aureus enterotoxin gene sequence for coding wild type staphylococcus aureus enterotoxin protein or a mutant type staphylococcus aureus enterotoxin gene sequence for coding mutant type staphylococcus aureus enterotoxin protein is led into the BCG viable bacterium strain. The invention further provides the construction method of the recombination strain and the viable bacterium vaccine obtained through the construction method. The viable bacterium strain can express and secrete the staphylococcus aureus enterotoxin protein in cells, and zoology experiments show that the function for prevention and treatment of cancers can be achieved after the viable bacterium strain is used as a vaccination organism.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant BCG live bacteria strain and live bacteria vaccine capable of expressing and secreting Staphylococcus aureus enterotoxin protein constructed and obtained by using genetic engineering technology, and a construction method and application thereof. Background technique [0002] Cancer is a killer that threatens human health and life, and the number of deaths caused by malignant tumors has risen to the top of various diseases every year. Currently, the main treatments for cancer are surgery, radiotherapy and chemotherapy. Although medical scientists continue to improve these three major treatment methods, many cancer patients are still difficult to cure. Traditional surgery, radiotherapy and chemotherapy have limited effects because the tumor itself is easy to metastasize and recur. At present, tumor immunotherapy has become a relatively effective treatment method. Immun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75A61K48/00A61P35/00C12R1/07
Inventor 胡章立李勇
Owner SHENZHEN UNIV
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