A kind of recombinant BCG and its construction and application

Abstract

The invention belongs to the field of medicine and health, and more particularly relates to a recombinant BCG (bacille calmette-guerin) vaccine as well as construction and application thereof. A knocked-out BCG strain after an autophagy inhibition gene in BCG is knocked out is taken as a recombinant BCG vaccine, and compared with wild BCG, the constructed recombinant BCG vaccine can obviously induce autophagy of macrophages and reduce the survival rate in infected macrophages, and can enhance antigen presentation and activate CD4<+>Th1 type reaction by autophagy pathway, so that the technical problems that the existing BCG vaccine is weak in protection and cannot effectively prevent tuberculosis of teenagers and adults due to the autophagy inhibition genes are solved.

Description

technical field

[0001] The invention belongs to the field of medicine and health, and more specifically relates to a recombinant BCG and its construction and application. Background technique

[0002] Although short-course combined chemotherapy under direct observation and BCG, the only preventive vaccine at present, have been used to treat and prevent tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) infection, respectively, in 2018, the global With tens of millions of new cases and 1.6 million deaths, TB is the number one killer of all infectious diseases. Therefore, it is still necessary to vigorously develop new treatment and prevention technologies to prevent and treat TB more effectively. Neonatal immunization with BCG has been included in the immunization strategy of more than 150 countries and regions around the world for the prevention of TB. Current research generally believes that although BCG immunization can protect infants and young children again...

Examples

Embodiment 1

[0049] BCG_2432c was detected in 13 strains of BCG worldwide:

[0050] (1) Primer design: F: 5'-TGCTCTAGACGCTCAAGCCGCTGCTGTCAGTC-3', containing the Xba I restriction site, covering the upstream 385 bp of the open reading frame; R-5'-CCCAAGCTTAGCTGCCGAGCCATGTTCAGTCC-3' containing the HindIII restriction site; covering the open reading frame 893 bp downstream of the box;

[0051] (2) PCR amplification of the target band, the system is 50 μl system:

[0052]

[0053] The PCR conditions are: 95°C for 5 minutes; 98°C for 5 seconds, 60°C for 5 seconds, 72°C for 2 minutes and 45 seconds, 29 cycles; 72°C for 10 minutes, 4°C forever.

[0054] (3) Wash the PCR product with a clean up kit, and use Xba I and HindIII to digest at 37°C for 30 minutes, the system is 30 μl

[0055]

[0056] (4) Digest the pcDNATM3.1 / myc-His(-)A plasmid with Xba I and HindIII at 37°C for 30 minutes, the system is 30 μl

[0057]

[0058] Use T4 ligase to ligate the digested PCR fragment and the vect...

Embodiment 2

[0064] Primer design for knockout of BCG_2432c

[0065] The left arm primers are: LFP5'-TTTTTTTTCCATAAATTGGCGCTCAAGCCGCTGCTGTCA-3', LRP5'-TTTTTTTTCCATTTCTTGGCCATCCCGACCACCTCAGAACC-3'; covering BCG_2432c open reading frame 184bp; primer design Van91I restriction site.

[0066] The right arm primers are: RFP5'-TTTTTTTTCCATAGATTGGTGCCAGTGATGTTCCCGTCCA-3', LRP5'-TTTTTTTTCCATCTTTTGGAGCTGCCGAGCCATGTTCA-3'; covering 40 bp of the open reading frame of BCG_2432c; the primers were designed with Van91I enzyme cutting site.

Embodiment 3

[0068] Amplify the left and right arms by PCR, and the PCR system is a 50 μl system:

[0069]

[0070] The PCR conditions are:

[0071] 95°C for 5 minutes; 95°C for 45 seconds, 58°C for 45 seconds, 72°C for 1 minute, cycle 29 times; 72°C for 1 minute, 4°C forever.

[0072] After the PCR reaction was completed, 1% agarose gel electrophoresis was performed to observe and the PCR product was recovered with a DNA gel recovery kit (AXYGEN) for future use.

technical field

[0001] The invention belongs to the field of medicine and health, and more specifically relates to a recombinant BCG and its construction and application. Background technique

[0002] Although short-course combined chemotherapy under direct observation and BCG, the only preventive vaccine at present, have been used to treat and prevent tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) infection, respectively, in 2018, the global With tens of millions of new cases and 1.6 million deaths, TB is the number one killer of all infectious diseases. Therefore, it is still necessary to vigorously develop new treatment and prevention technologies to prevent and treat TB more effectively. Neonatal immunization with BCG has been included in the immunization strategy of more than 150 countries and regions around the world for the prevention of TB. Current research generally believes that although BCG immunization can protect infants and young children again...

Examples

Embodiment 1

[0049] BCG_2432c was detected in 13 strains of BCG worldwide:

[0050] (1) Primer design: F: 5'-TGCTCTAGACGCTCAAGCCGCTGCTGTCAGTC-3', containing the Xba I restriction site, covering the upstream 385 bp of the open reading frame; R-5'-CCCAAGCTTAGCTGCCGAGCCATGTTCAGTCC-3' containing the HindIII restriction site; covering the open reading frame 893 bp downstream of the box;

[0051] (2) PCR amplification of the target band, the system is 50 μl system:

[0052]

[0053] The PCR conditions are: 95°C for 5 minutes; 98°C for 5 seconds, 60°C for 5 seconds, 72°C for 2 minutes and 45 seconds, 29 cycles; 72°C for 10 minutes, 4°C forever.

[0054] (3) Wash the PCR product with a clean up kit, and use Xba I and HindIII to digest at 37°C for 30 minutes, the system is 30 μl

[0055]

[0056] (4) Digest the pcDNATM3.1 / myc-His(-)A plasmid with Xba I and HindIII at 37°C for 30 minutes, the system is 30 μl

[0057]

[0058] Use T4 ligase to ligate the digested PCR fragment and the vect...

Embodiment 2

[0064] Primer design for knockout of BCG_2432c

[0065] The left arm primers are: LFP5'-TTTTTTTTCCATAAATTGGCGCTCAAGCCGCTGCTGTCA-3', LRP5'-TTTTTTTTCCATTTCTTGGCCATCCCGACCACCTCAGAACC-3'; covering BCG_2432c open reading frame 184bp; primer design Van91I restriction site.

[0066] The right arm primers are: RFP5'-TTTTTTTTCCATAGATTGGTGCCAGTGATGTTCCCGTCCA-3', LRP5'-TTTTTTTTCCATCTTTTGGAGCTGCCGAGCCATGTTCA-3'; covering 40 bp of the open reading frame of BCG_2432c; the primers were designed with Van91I enzyme cutting site.

Embodiment 3

[0068] Amplify the left and right arms by PCR, and the PCR system is a 50 μl system:

[0069]

[0070] The PCR conditions are:

[0071] 95°C for 5 minutes; 95°C for 45 seconds, 58°C for 45 seconds, 72°C for 1 minute, cycle 29 times; 72°C for 1 minute, 4°C forever.

[0072] After the PCR reaction was completed, 1% agarose gel electrophoresis was performed to observe and the PCR product was recovered with a DNA gel recovery kit (AXYGEN) for future use.

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