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Co-expression system and construction method of polyvalent bacteriophage lyase genes, live vaccine of carrying system and preparation and application of live vaccine

A phage lytic enzyme and co-expression technology, applied in the field of genetic engineering

Inactive Publication Date: 2015-06-03
伊正君
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary bacteria usually die after their cell walls are destroyed, but Mtb is an intracellular parasite, and some cell wall-deficient Mtb (cell wall deficient forms, CWD-form or L- form) may still survive for a long time

Method used

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  • Co-expression system and construction method of polyvalent bacteriophage lyase genes, live vaccine of carrying system and preparation and application of live vaccine
  • Co-expression system and construction method of polyvalent bacteriophage lyase genes, live vaccine of carrying system and preparation and application of live vaccine
  • Co-expression system and construction method of polyvalent bacteriophage lyase genes, live vaccine of carrying system and preparation and application of live vaccine

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Embodiment 1

[0060] The co-expression system of the three phage lyase genes uses the eukaryotic expression plasmid pZM0 as the expression vector, and inserts the LysinA, LysinB and Holin gene fragments into the pZM0 plasmid framework containing the Mycobacterium OriM replicon, so that the recombinant plasmid It also carries LysinA, LysinB and Holin lyase genes, and expresses three phage lyase genes (pZM0A) targeting Mycobacterium tuberculosis. The schematic diagram of the main frame structure of pZM0A is shown in figure 1 .

Embodiment 2

[0062] The co-expression system of three phage lyase genes uses the eukaryotic expression plasmid pZM0 as the expression vector, and inserts the LysinA gene fragment and the LysinB-link-Holin fusion gene fragment into the pZM0 plasmid framework containing the OriM replicon of mycobacteria In this method, the recombinant plasmid simultaneously carries LysinA, LysinB and Holin lyase genes, and expresses three kinds of phage lyase genes (pZM0A) targeting Mycobacterium tuberculosis simultaneously. The schematic diagram of the main frame structure of pZM0A is shown in figure 1 .

Embodiment 3

[0064] Construction expresses Mtb bacteriophage LysinA, the eukaryotic expression vector pZM0A of LysinB-link-Holin, the main frame structure schematic diagram of pZM0A ( figure 1 ):

[0065] Genomic DNA of D29 bacteriophage was extracted according to the instructions of the viral genomic DNA extraction kit (a kit produced by Dalian TaKaRa Company). The upstream primers and downstream primers were designed according to the LysinA, LysinB and Holin coding sequences of D29 bacteriophage registered in GenBank, and were synthesized by Dalian TaKaRa Company.

[0066] Upstream of LysinA: 5'-CATCCATGGGCGGCCGCATGACGCTCATAGTCACAC-3'

[0067] Downstream of LysinA: 5'-GATCTCGAGTTCGAATCATAGGGCTCCATTC-3'

[0068] Upstream of LysinB: 5'-CTACCATGGGCAAGCTTATGAGCAAGCCCTGGCT-3'

[0069] Downstream of LysinB: 5'-GAAGCGGCCGCTCTAGATCAGATCTGTCGTAGGAACTCG-3'

[0070] Upstream of Holin: 5'-CATCCATGGGCAAGCTTATGAGCCCCAAGATCCG-3'

[0071] Downstream of Holin: 5'-GTAGCGGCCGCTCTAGATCATCGGTTCCAGGGCT-3...

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Abstract

The invention discloses a co-expression system and construction method of polyvalent bacteriophage lyase genes, a live vaccine of a carrying system and preparation and application of the live vaccine. Eukaryotic expression plasmids are used as an expression vector, and LysinA, LysinB and Holin gene segments are directionally inserted into the plasmids to simultaneously express the co-expression system of the bacteriophage lyase genes of three kinds of targeted mycobacterium tuberculosis. Mycobacterium smegmatis with good targeting property of macrophages or genetically-modified recombinant BCG is used as a live vector, the co-expression system simultaneously carrying three kinds of genes is electrically transformed into the mycobacterium smegmatis or the genetically-modified recombinant BCG, and then the recombinant therapeutic tuberculosis live vaccine is obtained through expansion in vitro. The live vaccine has a good effect on the field of curing active tuberculosis or latent tuberculosis infection caused by proliferative mycobacterium tuberculosis, dormant mycobacterium tuberculosis and drug-resistant mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a phage lyase gene targeting Mycobacterium tuberculosis, and more specifically to a co-expression system of polyphage lyase genes, a live tuberculosis vaccine technology carrying the co-expression system and applications thereof. Background technique [0002] One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and about 90% of active tuberculosis develops from latent tuberculosis infection (LTBI). Mtb in LTBI patients is in a latent state, and the body cannot completely remove it; under certain conditions, Mtb can re-replicate, develop into active tuberculosis and produce clinical symptoms, and become a new source of tuberculosis infection. LTBI is closely related to the formation of persistent infection, persistent infection, relapse, extrapulmonary dissemination, and multidrug resistance of tuberculosis. [0003] At present, about...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/60A61K48/00A61K39/04A61K38/51A61P31/06
Inventor 伊正君付玉荣
Owner 伊正君
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