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Mosaic type DNA (Deoxyribonucleic Acid) vaccine pVAXI-Hsp 70/CD 80 for preventing and immunologically treating tuberculosis

A DNA vaccine and chimeric technology, applied in the field of tuberculosis vaccines and chimeric DNA molecules, can solve the problems of people who cannot improve the protection level of immunity, continued contact, and poor adult effects

Inactive Publication Date: 2014-03-26
钟森 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BCG immune failure is also related to the following factors (Baldwin SL, D, Souza C, Roberts A D, et al. Evaluation of new vaccines in the mouse and guinea pigmodel of tuberculosis. Infect Immun 1998,66(6):2951-2959): (1) BCG cannot improve the protection level of people who already have immunity due to other factors, (2) as individuals grow older, they may continue to be exposed to mycobacterial antigens in the surrounding environment, thereby pushing the immune response from Th1 to Th2 type (this is the main reason why BCG is effective in preventing tuberculosis in children but not effective in adults)

Method used

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  • Mosaic type DNA (Deoxyribonucleic Acid) vaccine pVAXI-Hsp 70/CD 80 for preventing and immunologically treating tuberculosis
  • Mosaic type DNA (Deoxyribonucleic Acid) vaccine pVAXI-Hsp 70/CD 80 for preventing and immunologically treating tuberculosis
  • Mosaic type DNA (Deoxyribonucleic Acid) vaccine pVAXI-Hsp 70/CD 80 for preventing and immunologically treating tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction, identification and biological activity of the pVAXI-Hsp70 / CD80 vector of the present invention

[0032] (1) Materials and reagents:

[0033] 1.1 Materials and equipment

[0034] The pVAXI plasmid g was obtained from Invitrogen Company; the LIPOFECTAMINE2000 liposome transfection reagent was obtained from Invitrogen Company; the eukaryotic expression of pcDNA3.1-Hsp70 / CD80 was constructed and preserved in the previous stage of the laboratory, and it could also be constructed according to the records of the prior art (Chinese Patent ZL200410001785.0, publication number CN1562362); Competent cells E.coli DH5α were purchased from Invitrogen; human embryonic kidney cells 293T were purchased from the Cell Bank of the Chinese Academy of Sciences; 20 SPF-grade female healthy BALB / c mice were purchased from the Third Military Medical University ; Restriction enzymes HindIII, XbaI, T4 ligase were purchased from New England Biolabs (NEB Company); DNA fragm...

Embodiment 2

[0042] Example 2. Distribution and expression of PVAX1-Hsp70 / CD80 in mice

[0043] 1. Animal experiment Twenty inbred BALB / c mice were raised in an individually ventilated IVC system. The animals were divided into 5 groups according to the injection time (3 animals in each group): 7-day group, 15-day group, 30-day group, 180-day group and negative control group. At each time point, 125 μl of chimeric vaccine and 7.5 g / L bupivacaine suspension (4:1), containing 100 μg of plasmid, were injected into the quadriceps muscle of mice. The control group was injected with 125 μl of normal saline. At 7, 15, 30 and 180 days after the injection, eyeball blood as well as muscle, lymph node, bone marrow, spleen, liver, lung, kidney and thymus tissues were collected and frozen at -80°C for future use.

[0044] 1.2.4.2 RT-PCR detection of Hsp70 and CD80 expression in mice Extract RNA from muscle, lymph node, bone marrow, spleen, liver, lung, kidney, thymus tissue, according to Tiangen total...

Embodiment 3

[0065] Example 3 Study on the Immunoprotective Effect and Mechanism of pvAXI-Hsp70 / CD80 Chimeric DNA Vaccine on Tuberculosis

[0066] (1) Test method.

[0067] 1. Experimental materials:

[0068] Endotoxin-free plasmid purification system (Qiagen). IFN-γ, IL-4, (Miltenyi Biotec).

[0069] Preparation of DNA plasmids: Preparation of pVAXI-Hsp70 / CD80 and PcDNA with an endotoxin-free plasmid purification system 3.1 - Hsp70 / CD80 plasmid.

[0070] 2. Experiment group modeling and processing:

[0071] Animal grouping: random grouping according to the random number table, the experimental animals were 6-8 week old, clean grade healthy mice C57BL / 6N, weighing about 18±2g, half male and half male, purchased from the Experimental Animal Center of Chongqing Medical University. Divided into blank control group (referred to as control group), BCG vaccine group (BCG group, referred to as BCG group), tuberculosis experimental model group (referred to as tuberculosis group), pcDNA3.1-Hsp...

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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a tubercular mosaic type DNA (Deoxyribonucleic Acid) vaccine. The tubercular mosaic type DNA vaccine solves the technical problems of nonideal medicine effect and bad safety of the traditional mosaic vaccine. The technical scheme is as follows: the mosaic type DNA vaccine contains the encoding gene of HSP 70 / CD 80 mosaic protein and can express the encoding gene in vivo. After using a pVAXI carrier, the tubercular mosaic type DNA vaccine disclosed by the invention has a better effect on the prevention and treatment of tuberculosis and can especially be used for inducing stronger Th1 type reaction and better prevent and treat tuberculosis; and besides, the pVAXI carrier has kanamycin resistance, cannot cause any anaphylactic reaction and can better prevent and immunologically treat tuberculosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a tuberculosis vaccine, in particular to a chimeric DNA molecule capable of inducing and enhancing protective immune responses (humoral immunity and cellular immunity) in humans or animals. The DNA molecule can be used as a vaccine for the prevention and treatment of tuberculosis. Background technique [0002] Following the first and second world wars, there were two peaks of tuberculosis recovery in the world. In the mid-1980s, the third recovery peak was formed due to the sharp deterioration of the worldwide tuberculosis epidemic. According to the "World Health Organization Bulletin", in 1996 In 2010, the number of deaths from all ages in the world, tuberculosis ranks fourth after ischemic heart disease, cerebrovascular disease and acute lower respiratory infection, and ranks first in the number of deaths caused by a single disease in the world. The highest record in history. The num...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/04A61P31/06C12N15/62C12N15/63C07K19/00
Inventor 钟森史小玲
Owner 钟森
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