Recombination BCG viable bacterium strain capable of expressing and secreting human p53 protein, viable bacterium vaccine and construction method and application thereof

A technology of live bacteria and strains, applied in the field of recombinant BCG live bacteria strains, can solve the problems that cancer patients are difficult to be cured, the effects of radiotherapy and chemotherapy are limited, and tumors are easy to metastasize

Active Publication Date: 2014-01-08
深圳市微宇生物科技有限公司
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  • 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 tu

Method used

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  • Recombination BCG viable bacterium strain capable of expressing and secreting human p53 protein, viable bacterium vaccine and construction method and application thereof
  • Recombination BCG viable bacterium strain capable of expressing and secreting human p53 protein, viable bacterium vaccine and construction method and application thereof
  • Recombination BCG viable bacterium strain capable of expressing and secreting human p53 protein, viable bacterium vaccine and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Gene modification of human p53 and upstream addition of BCG secretion signal peptide sequence

[0035]According to the human p53 gene sequence (GenBank accession number: NM_001126112) and BCG genome codon preference, the human p53 preferred gene sequence suitable for BCG expression was designed and synthesized artificially. The preferred human p53 gene sequence is shown in SEQ ID NO:2.

[0036] By consulting relevant literature, the 120bp α-secretion signal peptide gene sequence carried by the BCG genome was obtained, such as the nucleotide sequence shown in SEQ ID NO:3. According to the restriction site requirements of the shuttle expression vector pMN234, the α-secretion signal peptide gene sequence was spliced ​​into the upstream of the human p53 preferred gene, BamH I and Xba I restriction sites were added upstream of the α-secretion signal peptide gene sequence, and the downstream of the human p53 preferred gene Add HindIII restriction site. The α-p53 gene sequen...

Embodiment 2

[0038] Construction of BCG Shuttle Expression Vector

[0039] 1. Construction of pMN-p53 shuttle expression vector

[0040] Plasmid pMN234 is an Escherichia coli-Mycobacterium shuttle expression vector, which also contains the HSP60 promoter (gifted by Beijing Proteomics Research Center). The target gene can be inserted between the BamH I and Hind III restriction sites of the pMN234 vector to express the target protein. In Example 1, the human p53 preferred gene containing the α secretion signal peptide was subcloned upstream, and the recombinant plasmid pUC-α-p53 contained BamH I and Hind III restriction sites at both ends).

[0041] (1) Extract pMN234 empty plasmid and clone plasmid pUC-α-P53

[0042] Rapid mini-extraction of plasmid DNA: Inoculate Escherichia coli strains containing pMN234 empty plasmid and cloned plasmid pUC-α-P53 respectively in 30ml LB medium containing kanamycin resistance and ampicillin resistance, shake at 37°C and 200rpm Incubate overnight. Plasm...

Embodiment 3

[0066] Genetic Transformation of BCG Shuttle Expression Vector

[0067] BCG was cultured in M7H9+ADC medium to the logarithmic growth phase. After pre-cooling on ice, centrifuge at 6000rpm for 5 minutes to collect BCG cells, wash the cells three times with 10% pre-cooled sterilized glycerol, and finally Suspended with 10% glycerol, prepared into BCG electroporation transformation competent cells.

[0068] Extract the pMN-p53 shuttle expression secretion vector plasmid (the method is the same as before), 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 cuvette, add 5 μl of shuttle expression secretion vector plasmid pMN-p53 with a concentration of 10 μg / ml, mix gently and place on ice for 10 min. 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 medium to ...

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Abstract

The invention provides a recombination BCG viable bacterium strain capable of expressing and secreting human p53 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 being used for prevention and treatment of human 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 human p53 optimization gene sequence obtained after the preference of a codon is modified by a human p53 gene sequence for coding the human p53 protein referring to a BCG genome 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 human p53 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 human p53 protein, a live bacterial vaccine constructed by 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 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 immunotherapy includes...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75A61K48/00A61P35/00C12R1/07
Inventor 胡章立李勇
Owner 深圳市微宇生物科技有限公司
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