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Recombinant bcg live bacterial strain expressing and secreting Staphylococcus aureus enterotoxin protein, live bacterial vaccine and its construction method and application

A technology of Staphylococcus aureus enterotoxin and live bacteria, applied in the biological field, can solve the problems that cancer patients are difficult to be cured, tumors are easy to transfer, and the effects of radiotherapy and chemotherapy are limited.

Active Publication Date: 2016-02-10
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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  • Recombinant bcg live bacterial strain expressing and secreting Staphylococcus aureus enterotoxin protein, live bacterial vaccine and its construction method and application
  • Recombinant bcg live bacterial strain expressing and secreting Staphylococcus aureus enterotoxin protein, live bacterial vaccine and its construction method and application
  • Recombinant bcg live bacterial strain expressing and secreting Staphylococcus aureus enterotoxin protein, live bacterial vaccine and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Detoxification mutation transformation of wild-type Staphylococcus aureus enterotoxin SEA and SEC2 genes

[0035] According to related literature reports (VladimirS, etal.Toxicon.2004, 43:433-438., etc.) SEA and SEC2 gene sequences (GenBank accession number: NM_001126112) were genetically modified, and the modified sites were the amino acid sites of SEA (D227A) and SEC2 (T20L; T20L / G22E; G22E / N23A; T20L / G22E / N23A).

[0036] 1. Design primers

[0037] Utilize GeneTool software to design related 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) Primers for single site mutation of SEC2 gene T20L

[0044] Forward:

[0045] 5'-tttaatgggtaatatgaaatatttatatgatgatca-3'

[0046] Reverse:

[0047] 5'-ccagtaaactcacttgatttgtgcaactc-3'

[0048] (3) SEC2 gene T20L / G22E double s...

Embodiment 2

[0089] Construction of BCG Shuttle Expression Vector

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

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

[0092] Plasmid pMN234 is an Escherichia coli-mycobacterium shuttle expression vector, which also contains the HSP60 promoter.

[0093] Introduction of α-secretion signal peptide:

[0094] The pMN234 plasmid does not contain an α-secretion signal peptide sequence. In order to allow the target protein to be secreted and expressed in mycobacteria, we inserted a mycobacterium α-secretion signal peptide sequence downstream of the pMN234 vector promoter, which has the nucleotides shown in SEQ ID NO:11 acid sequence.

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

[0096] Forward: 5'-CGC GGATCC ATGACAGACGTGAG-3'

[0097] Un...

Embodiment 3

[0128] Genetic Transformation of BCG Shuttle Expression Vector

[0129] BCG was cultured in the M7H9+ADC medium to the logarithmic growth phase. After pre-cooling on ice, centrifuge at 6000rmp for 5 minutes to collect BCG bacterial cells, wash the bacterial cells three times with 10% pre-cooled sterilized glycerol, and finally Suspended with 10% glycerol, prepared into BCG electroporation transformation competent cells.

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

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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 bacterial strain capable of expressing and secreting Staphylococcus aureus enterotoxin protein constructed by genetic engineering technology, a live bacterial vaccine 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 from malignant tumors has risen to the top of various diseases every year. At present, the main treatment methods for tumors are surgery, radiotherapy and chemotherapy. Although medical scientists continue to improve these three major treatment methods, many cancer patients are still difficult to be cured. Because the tumor itself is prone to metastasis and recurrence, the effects of traditional surgery, radiotherapy and chemotherapy are limited. At present, tumor immunotherapy has become a relatively effective treatment method. Tumor...

Claims

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Application Information

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