Recombinant human co-stimulatory molecule bacilli-calmette-guerin strain and process for making same

Abstract

The invention discloses a recombinant human co-stimulatory molecule bacilli-calmette-guerin strain and process for preparation, wherein the cultrue preservation number is CGMCC No.1120. The preparing process consists of carrying out hB7-2(IgC+IgV) fragment polymerase chain reaction, establishing plasmid pYL-hB7-2 and transforming, carrying out E.Coli-pYL-hB7-2 monoclonal bacterial colony expansion, plasmid extraction and purification, electrophoresis, enzyme cutting and determination of pYL-hB7-2 plasmid through PCR reaction, bacillus Calmette-Guerin vaccine electrical transformation, rBCG-hB7-2 monoclonal bacterial colony selection, expansion, and determination.

Description

technical field

[0001] The invention relates to genetic engineering technology, in particular to a recombinant human co-stimulatory molecule Bacillus Calmette-Guerin strain and a preparation method thereof. Background technique

[0002] Bacillus Calmette-Guerin (BCG) is a highly virulent biological immunomodulator formed by attenuated Mycobacterium bovis, which has a high degree of safety and few serious complications, and is widely used in the prevention of tuberculosis all over the world.

[0003] Intravesical infusion of wild-type BCG has achieved satisfactory clinical efficacy in the prevention of tumor recurrence, treatment of residual tumors and carcinoma in situ, but 30-45% of patients still do not respond to intracavitary BCG infusion therapy. Endo-BCG perfusion can cause local and systemic reactions in the bladder, 5% of patients may have serious complications, and 0.5% may even be life-threatening, which limits the prevention and treatment of superficial bladder tu...

Examples

Embodiment Construction

[0024] 1. Materials

[0025] (1) Bacterial strain: Escherichia coli is DH5α, which is preserved in our institute. BCG was purchased from Beijing Institute of Biological Products, Denmark type I.

[0026] (2) Plasmid: The vector plasmid pYL-hIFN-α-2B plasmid was provided by Professor YI LUO of Iowa University, USA. The huB7-2(IgV+C) / pGEX-4T-3 plasmid containing hB7-2 cDNA was provided by Mr. Lai Baochang from Xi'an Medical University.

[0027] (3) Main instruments and equipment

[0028] 1. Microplate reader: SUNRISE TECAN A-5082 Austria

[0029] 2. Constant temperature incubator and desktop centrifuge:

[0030] 3. Low temperature and high speed centrifuge: BECKMAN J2-HS USA

[0031] 4. PCR instrument: PERKIN ELEMER GeneAmp PCR System 2400 USA

[0032] 5. Electroporator: Genepulser Electroprotocol, USA

[0033] (4) Preparation of main reagents and solutions

[0034] 1. Gel Extraction Kit: E.Z.N.A Gel Extraction Kit

[0035] 2. Purification Kit: TaKaRa DNA Fragment Purif...

technical field

[0001] The invention relates to genetic engineering technology, in particular to a recombinant human co-stimulatory molecule Bacillus Calmette-Guerin strain and a preparation method thereof. Background technique

[0002] Bacillus Calmette-Guerin (BCG) is a highly virulent biological immunomodulator formed by attenuated Mycobacterium bovis, which has a high degree of safety and few serious complications, and is widely used in the prevention of tuberculosis all over the world.

[0003] Intravesical infusion of wild-type BCG has achieved satisfactory clinical efficacy in the prevention of tumor recurrence, treatment of residual tumors and carcinoma in situ, but 30-45% of patients still do not respond to intracavitary BCG infusion therapy. Endo-BCG perfusion can cause local and systemic reactions in the bladder, 5% of patients may have serious complications, and 0.5% may even be life-threatening, which limits the prevention and treatment of superficial bladder tu...

Examples

Embodiment Construction

[0024] 1. Materials

[0025] (1) Bacterial strain: Escherichia coli is DH5α, which is preserved in our institute. BCG was purchased from Beijing Institute of Biological Products, Denmark type I.

[0026] (2) Plasmid: The vector plasmid pYL-hIFN-α-2B plasmid was provided by Professor YI LUO of Iowa University, USA. The huB7-2(IgV+C) / pGEX-4T-3 plasmid containing hB7-2 cDNA was provided by Mr. Lai Baochang from Xi'an Medical University.

[0027] (3) Main instruments and equipment

[0028] 1. Microplate reader: SUNRISE TECAN A-5082 Austria

[0029] 2. Constant temperature incubator and desktop centrifuge:

[0030] 3. Low temperature and high speed centrifuge: BECKMAN J2-HS USA

[0031] 4. PCR instrument: PERKIN ELEMER GeneAmp PCR System 2400 USA

[0032] 5. Electroporator: Genepulser Electroprotocol, USA

[0033] (4) Preparation of main reagents and solutions

[0034] 1. Gel Extraction Kit: E.Z.N.A Gel Extraction Kit

[0035] 2. Purification Kit: TaKaRa DNA Fragment Purif...

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