Preparation method of Burkholderia pseudomallei recombined BLF1 protein, product prepared through preparation method and application of Burkholderia pseudomallei recombined BLF1 protein

A Kkholderia, protein technology, applied in the preparation of Burkholderia pseudomallei recombinant BLF1 protein, Burkholderia pseudomallei recombinant BLF1 protein and the application of the protein, can solve the problem of pathogenic mechanism Clear and other issues, to achieve the effect of easy separation and purification, simple steps, and high expression

Inactive Publication Date: 2016-04-20
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogen of this disease is Burkholderia pseudomallei (Burkholderia pseudomallei, BP), which belongs to Gram-negative bacilli, is widely distributed, mainly exists in water or soil, and is directly infected through mucous membrane adhesion and wound surface in the form of aerosol or bacteria-carrying liquid. The body, after entering the body, is swallowed by natural immune cells, but it can cause abscess through a series of escape mechanisms, leading to disease
[0003] At present, the pathogenic mechanism of Burkholderia pseudomallei is still unclear, and it is highly resistant to antibiotics. There is no effective vaccine against melioidosis, but virulence is an important factor that causes disease after pathogenic bacteria invade the body.

Method used

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  • Preparation method of Burkholderia pseudomallei recombined BLF1 protein, product prepared through preparation method and application of Burkholderia pseudomallei recombined BLF1 protein
  • Preparation method of Burkholderia pseudomallei recombined BLF1 protein, product prepared through preparation method and application of Burkholderia pseudomallei recombined BLF1 protein
  • Preparation method of Burkholderia pseudomallei recombined BLF1 protein, product prepared through preparation method and application of Burkholderia pseudomallei recombined BLF1 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, Cloning of the full-length fragment of the Bacillus pseudomallei virulence factor BLF1 gene

[0030] Firstly, according to the full-length gene sequence of BPK96243BLF1 protein, the PCR method was used to amplify the BLF1 target gene fragment from the genome of Burkholderia pseudomallei BPC006, and the amplification steps were as follows:

[0031] Design PCR primers as follows, respectively SEQIDNO.1 and SEQIDNO.2 (the underline indicates the base sequence of the restriction site);

[0032] BLF1-F: 5'-cg ggatcc atgcccaactcactcgaag-3' (SEQ ID NO: 1), the underline is the BamHI restriction site;

[0033] BLF1-F: 5'-ccg ctcgagctattgcttgcgctgctg-3' (SEQ ID NO: 2), the underline is the XhoI restriction site;

[0034] Take out the preserved Burkholderia pseudomallei BPC006 strain from the -80°C freezer and spread it on the BPC006 special solid medium, culture it overnight at 37°C, and then pick a single colony and inoculate it in the BPC006 special liquid medi...

Embodiment 2

[0059] Example 2, BLF1 induced expression purification and antigen preparation in prokaryotic expression system-Escherichia coli

[0060] 1. Induced expression of target protein

[0061] 1) Take the correctly identified pGEX-6P-2 / BLF1 bacterial solution and culture it to OD 600 When the temperature is 0.6, take out 200 μl of the bacterial solution, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, resuspend the bacterial cells in 40 μl of PBS buffer, add 10 μl of 5× protein loading buffer, boil at 100°C for 5 minutes, and store in the refrigerator at 4°C for later use; the remaining bacterial solution IPTG was added to make the final concentration 0.2mmol / L, and placed on a shaker at 30°C to induce expression for 3h.

[0062] 2) Take out the bacterial solution after induced expression: take out 100 μl of bacterial solution, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, resuspend the bacterial cells in 40 μl of PBS buffer, add 10 μl of 5× protein ...

Embodiment 3

[0071] Embodiment 3, the detection of immune animal and antibody

[0072] 1. Immunization of animals

[0073] 1) For the first immunization, use PBS to dilute BLF1 protein to 1mg / mL and grind it with complete Freund's adjuvant at a volume ratio of 1:1 until it becomes milky. Use a 2.5mL syringe to treat the neck, back, and bilateral groin of New Zealand white rabbits. Perform subcutaneous immunization and set up a negative control group;

[0074] 2) The second immunization was carried out 7 days later, using incomplete Freund's adjuvant and BLF1 protein with a concentration of 1 mg / mL to grind at a volume ratio of 1:1, and the immunization method was the same as above;

[0075] 3) Seven days after the second immunization, the third immunization was carried out, and the immunization route was the same as that of the second immunization;

[0076] 2. Detection of IgG humoral response level of BLF1 protein by ELISA method

[0077] On the 14th day after the third immunization, t...

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Abstract

The invention discloses a preparation method of Burkholderia pseudomallei recombined BLF1 protein, the product prepared through the preparation method and an application of the Burkholderia pseudomallei recombined BLF1 protein. The preparation method includes the steps that a primer for amplifying a Burkholderia pseudomallei BLF1 gene is designed firstly, a sequence shown in SEQ ID NO.3 of a coding area sequence is obtained through amplification, then the acquired sequence is connected to an expression vector to construct a recombinant expression vector, the recombinant expression vector is converted into host bacteria, and through inducible expression, the Burkholderia pseudomallei recombined BLF1 protein is obtained. The preparation method is simple, the space design conception and immunogenicity of the purified protein can be kept to the maximum degree, the purity of the protein is larger than 95%, it is proved through a mouse experiment that the purified protein has animal toxicity, it is shown that the purified protein has biological activity, the purified protein acts on A549 cells, cell growth can be restrained, and thus the Burkholderia pseudomallei recombined BLF1 protein can be used for preparing anti-tumor medicine and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of Burkholderia pseudomallei recombinant BLF1 protein, and also relates to the Burkholderia pseudomallei recombinant BLF1 protein prepared by the method and the application of the protein. Background technique [0002] Melioidosis (Melioidosis) is a new infectious disease, mainly prevalent in tropical and subtropical regions, and my country's Guangdong, Taiwan, Hong Kong, and Hainan are also important endemic regions. The pathogen of this disease is Burkholderia pseudomallei (Burkholderia pseudomallei, BP), which belongs to Gram-negative bacilli, is widely distributed, mainly exists in water or soil, and is directly infected through mucous membrane adhesion and wound surface in the form of aerosol or bacteria-carrying liquid. The body, after entering the body, is swallowed by natural immune cells, but it can cause abscesses and diseases through a seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/195G01N33/68G01N33/569A61K38/16A61P35/00
CPCC07K14/195A61K38/00G01N33/56911G01N33/68
Inventor 任春艳毛旭虎方瑶李倩胡艺马腾飞胡志强
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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