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A kind of tuberculosis gene drug and preparation method thereof

A gene drug and tuberculosis technology, applied in the field of biomedicine, can solve problems such as diseases and curative effects that need to be improved, and achieve the effect of improving safety performance

Active Publication Date: 2017-11-14
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of the above-mentioned recombinant adenovirus is that it is only effective for the treatment of M.tb parasitic in the cells, but the free M.tb outside the cells can still cause disease
Therefore, the efficacy of the aforementioned recombinant adenovirus-based TB gene therapy drugs still needs to be improved.

Method used

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  • A kind of tuberculosis gene drug and preparation method thereof
  • A kind of tuberculosis gene drug and preparation method thereof
  • A kind of tuberculosis gene drug and preparation method thereof

Examples

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preparation example Construction

[0054] The TB gene medicine provided by the invention, its preparation method comprises the following steps:

[0055] Step 1, integrating the first sequence expressing intracellular granulysin protein and the second sequence expressing extracellular granulysin protein into the shuttle plasmid to form the intracellular granulysin gene shuttle plasmid (pDChGLi) and the extracellular Granlysin gene shuttle plasmids (pDChGLs);

[0056] The first sequence is preferably:

[0057] (1) the gene sequence shown by SEQ ID No.1; or

[0058] or

[0059] (3) The amino acid sequence defined by SEQ ID No.1 is increased, deleted or replaced by one or more amino acids, and the expression product has a gene sequence with the same activity as the protein expressed by the sequence SEQ ID No.1.

[0060] The second sequence is preferably:

[0061] A, the gene sequence shown by SEQ ID No.2; or

[0062] B. An amino acid sequence with an amino acid sequence homology defined by the sequence SEQ ID ...

Embodiment 1

[0072] Example 1 Packaging and purification of intracellular granulysin recombinant adenovirus (rAdhGLi) and extracellular granulysin recombinant adenovirus (rAdhGLs)

[0073] 1) According to conventional molecular cloning techniques, the gene sequences of SEQ ID No.1 and SEQ ID No.2 were respectively cloned into the adenovirus-Escherichia coli shuttle vector pDC316 to obtain recombinant plasmids pDChGLi and pDChGLs, which were confirmed by enzyme digestion and sequencing.

[0074] 2) HEK293 cells were co-transfected with plasmid pDChGLi (or pDChGLs) and backbone plasmid pBHGloxΔE1,3Cre (1:1) to package recombinant virus rAdhGLi (or rAdhGLs).

[0075] (1) Inoculate HEK293 cells in good growth state in a six-well plate, 5×10 per well 5 Cells were cultured in a 37°C, 5% CO2 cell incubator for 18-24 hours, so that the confluence of the cells reached about 90%. The culture medium was discarded, and 1 mL of complete DMEM medium without antibiotics was added to each well.

[0076]...

Embodiment 2

[0105] Example 2 RT-qPCR confirmed the expression of granulysin mRNA level after rAdhGLi or rAdhGLs infected cells

[0106] 1) AdNull, rAdhGLi and rAdhGLs were infected with HEK293 cells respectively

[0107] (1) HEK293T cells in a good state of trypsinization were resuspended by adding DMEM complete medium and counted at 5×10 5 The total number of cells was inoculated into each well of a six-well plate, supplemented with DMEM complete medium to 2ml, and placed in a 37°C CO2 incubator for 18-24h.

[0108] (2) Discard the old medium in each well, add 2ml of DMEM complete medium without antibiotics, and infect HEK293T with AdNull, rAdhGLi and rAdhGLs at an infection rate of MOI of 10, and culture for 72 hours.

[0109] (3) Collect the cell suspension with sterile 1.5ml EP, centrifuge at 1000rpm / min for 10min, discard the supernatant to collect the cell pellet, wash the cells twice with sterile PBS, discard the supernatant by centrifugation, add 1ml Trizol to each EP tube Repea...

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Abstract

The invention discloses a tuberculosis gene medicine and a preparation method thereof. The tuberculosis gene medicine takes an adenovirus as a gene expression vector for expressing intracellular granulysin protein and extracellular granulysin protein, wherein the expression quantity of the intracellular granulysin protein and the extracellular granulysin protein ranges from (2: 8) to (8: 2). The method comprises the following steps: preparing an intracellular granulysin protein gene shuttle plasmid and an extracellular granulysin protein gene shuttle plasmid; preparing a first adenovirus for expressing the intracellular granulysin protein and a second adenovirus for expressing the extracellular granulysin protein; mixing at the quantity ratio ranging from (2: 8) to (8: 2) to prepare the gene medicine provided by the invention. The gene medicine provided by the invention is administrated in one step through combining intracellular and extracellular granulysin recombinant adenoviruses; compared with independent utilization of the intracellular and extracellular granulysin recombinant adenoviruses, the tuberculosis gene medicine can be used for treating primary tuberculosis better, even multi-drug resistant tuberculosis.

Description

technical field [0001] The invention belongs to the field of biomedicine, and more specifically relates to a gene medicine for tuberculosis and a preparation method thereof. Background technique [0002] Adenovirus (Ad), as a common gene expression vector, can be used to mediate the expression of foreign genes in the body. At present, nearly 25% of clinical trials of gene drugs are based on recombinant adenovirus (rAd), showing its important status and safety in medicine; moreover, four gene drugs that have been approved for clinical use at home and abroad Among the vaccines and vaccines, the two approved by China are based on rAd, and their safety has been confirmed. The preparation of rAd is simple, the titer of the virus produced is high, and it can be applied in various ways such as respiratory inhalation, intramuscular injection and local injection. [0003] Tuberculosis (TB) is not only the number one killer of infectious diseases threatening global human health, but...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K38/19A61P31/06C12N15/861C12N7/01C12N15/19C07K14/52
CPCA61K38/19A61K48/005C07K14/52C12N15/86C12N2710/10043
Inventor 范雄林
Owner HUAZHONG UNIV OF SCI & TECH
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