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Mosaic type DNA vaccine in use for preventing tuberculosis and immunological therapy

A DNA vaccine and chimeric technology, applied in gene therapy, application, drug combination and other directions, can solve the problems of unable to improve the protection level of immune population and poor adult effect.

Inactive Publication Date: 2006-11-15
钟森 +3
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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 them and guinea pig model of tuberculosis. Infect. Immun. 1998, 66 (6): 2951-2959 .): (1) BCG cannot improve the level of protection 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 shifting the immune response from the Th1 type Push 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 vaccine in use for preventing tuberculosis and immunological therapy
  • Mosaic type DNA vaccine in use for preventing tuberculosis and immunological therapy
  • Mosaic type DNA vaccine in use for preventing tuberculosis and immunological therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] [Example 1] Construction of pcDNA3.1(+)-hsp70 / B7-1 chimeric plasmid.

[0039] see figure 1 with figure 2 :

[0040] main experimental materials

[0041] 1. Plasmids and bacterial strains: pcDNA3.1(+)-B7-1 (including human B7-1 coding gene and regulatory genes on both sides) and plasmid pcDNA3.1(+) were purchased from Invitrogen, and engineering bacteria Dh5α was purchased from Shanghai Sangong bioengineering company.

[0042]2. Design of oligonucleotide primers: According to the published gene sequence of human tuberculosis H37Rv strain hsp70 (Mckenzie.KR, Adams E, Britton WJ, et al.Sequence and immunogenicity of the 70-kDa heatshock protein of Mycobacterium leprae.J .Immune.1991;147.:312-319.) and human B7-1 sequence (GenBank accession number NM005191) design.

[0043] 1. Recombinant pcDNA3.1(+)-hsp70 plasmid primer:

[0044] P1 (SEQ ID NO.4): 5'ggagccATGGCTCGTGCGGTCGGGATC 3' (containing BamHI site and initiation codon ATG)

[0045] P2 (SEQ ID NO. 5): 5' gaattc...

Embodiment 2

[0111] [Example 2] Expression of pcDNA3.1(+)hsp70 / B7-1 chimeric DNA in eukaryotic cells

[0112] The pcDNA3.1(+)hsp70 / B7-1 chimeric DNA was encapsulated and transfected into eukaryotic cells (COS.7) with liposomes, and the mRNA was extracted for RT-PCR at 24.48.72 hours, and the product was subjected to agarose electrophoresis. See the target zone ( Image 6 ).

Embodiment 3

[0113] [Example 3] pcDNA3.1 (+) hsp70 / B7-1 chimeric DNA vaccine immune prevention effect research on mice

[0114] Animal prevention experiment: DNA vaccine inoculation: experimental animals 6-8 weeks old, clean grade healthy mouse C57BL / 6N (purchased from Animal Experiment Center of Chongqing Medical University). Eight rats in each group, half male and half male, were divided into experimental group and control group. The experimental group was injected with 100 μg of pcDNA.3.1(+), pcDNA.3.1(+)-hsp70 and pcDNA.3.1(+)-hsp70 / B7-1 respectively. (0.5ml), BCG was injected according to body weight; the control group was injected with normal saline 0.5ml. The injection site is the tibialis anterior muscle on both sides, and the dose is divided into half on each side. Inoculation 3 weeks and 6 weeks each according to the original plan booster vaccination once. Three weeks after the third immunization, each mouse was injected with H37RV tail vein for 10 4 -10 5 CFU.

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Abstract

The present invention relates to a chimeric DNA vaccine for preventing tuberculosis and immunotherapy, comprising a full-length open reading frame of the nucleotide sequence shown in SEQ ID NO.1, or a conservative variant thereof, in SEQ ID NO.1 It comprises mycobacterium hsp70 gene and human B7-1 gene, and a linker sequence is designed between the two genes to connect them. The vaccine of the invention can be used for tuberculosis caused by Mycobacterium tuberculosis; or express the corresponding protein in eukaryotic cells in vitro for preventing and treating tuberculosis. It can also be used as an immune enhancer for corresponding viral diseases and tumor immunotherapy.

Description

technical field [0001] The invention relates to a novel 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 is used as a vaccine for the prevention and treatment of tuberculosis; at the same time, as an immune enhancer, it can improve the immune function of humans or animals, and achieve the effect of preventing and treating viral diseases and tumors. 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 The number of deaths of all ages in the world in 2019, tuberculosis ranks fourth after the number of deaths from ischemic heart disease, cerebrovascular disease a...

Claims

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Application Information

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IPC IPC(8): C12N15/31A61K39/04A61K39/00A61K48/00A61P31/06A61P37/02
Inventor 钟森丁惠忠史小玲钟林
Owner 钟森
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