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Tuberculosis vaccine and preparation technology thereof

A preparation process, a technology for tuberculosis, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem that it is difficult to obtain the effect of stimulating the activation of specific lymphocytes, cannot kill and clear Mycobacterium tuberculosis, The problem of poor killing effect of latent infectious bacteria can achieve the effect of enhancing killing and clearing of tuberculosis bacteria, enhancing antigen-specific immune response, and improving antigen presentation ability.

Inactive Publication Date: 2018-10-09
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the tuberculosis vaccines in the prior art mainly exert anti-tuberculosis effects by activating lymphocytes, but tuberculosis mainly parasitizes in macrophages in the body. Although lymphocytes are considered to be the main effector cells of tuberculosis immunity, it is still necessary to It acts through macrophages, and a large number of studies have confirmed that Mycobacterium tuberculosis can inhibit the function of macrophages through a series of mechanisms, including sensitivity to external stimuli. Therefore, even if lymphocytes are well activated, it is still difficult to clear macrophages. The problem with intracellular tuberculosis
[0005] The existing tuberculosis chemotherapeutic drugs have a good killing and clearing effect on the active infection of sensitive Mycobacterium tuberculosis, but the main problem they face is that the killing effect on latent infectious bacteria is poor, which leads to the predicament that the current treatment of tuberculosis is too long
At present, the stimulating antigen used in most tuberculosis vaccines is the immunodominant antigen secreted by Mycobacterium tuberculosis in the early stage. Although it can stimulate a strong immune response, it cannot recognize Mycobacterium tuberculosis in a state of latent infection, so it cannot kill and treat mycobacterium tuberculosis. Clearing Mycobacterium tuberculosis in a latent infection state is of little clinical help
[0006] In recent years, there have also been a small number of reports on the use of latent infection-specific antigens for vaccine design. However, due to the existence of: (1) the activation of specific lymphocytes is also the main factor, which cannot kill and remove latent tuberculosis bacteria in macrophages; 2) Since Mycobacterium tuberculosis can inhibit the function of macrophages, the efficiency of vaccine antigen presentation to lymphocytes is poor, and it is difficult to obtain the desired effect of stimulating specific lymphocyte activation

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  • Tuberculosis vaccine and preparation technology thereof
  • Tuberculosis vaccine and preparation technology thereof
  • Tuberculosis vaccine and preparation technology thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] A tuberculosis vaccine designed on the basis of Figure 17 As shown, it includes a recombinant lentiviral expression plasmid pLenti-spHL47 / pLenti-spHI1 and lentiviral particles packaged with the recombinant lentiviral expression plasmid. The lentiviral particles are used to carry the above-mentioned lentiviral expression vectors, so that they can efficiently enter cells and express the above-mentioned recombinant target molecules for a long time.

[0056] Among them, pLenti-spHL47 / pLenti-spHI1 includes the following main components:

[0057] (1) Lentiviral framework plasmid: CS-CDF-CG-PRE. Function: used to provide frame information of lentiviral expression vector.

[0058] (2) Macrophage specific promoter: SP146. Function: This is a macrophage-specific promoter, which is used to control the expression of hspX gene and lrg47 / irgm1 gene, so that they can only be expressed in macrophages, but not in other cells.

[0059] (3) hspX gene: an expression gene that specific...

Embodiment 2

[0063] The preparation technology of tuberculosis vaccine of the present invention comprises the following steps:

[0064] 1. Using the plasmid pSP146-GFP as a template, the macrophage-specific promoter SP146 fragment was amplified by PCR technology (primers used: S1 / S2, the sequence is shown in Table 1), and then molecular cloning technology (refer to Sambrook.J editor-in-chief The third edition of "Molecular Cloning Experiment Guide"), the SP146 promoter was cloned into the lentiviral framework plasmid CS-CDF-CG-PRE using restriction enzymes EcoRI and AgeI to replace the CMV promoter in the original plasmid, Obtain macrophage-specific expression lentiviral expression vector pLenti-SP146.

[0065] 2. Use the PCR method (refer to the third edition of "Molecular Cloning Experiment Guide" edited by Sambrook.J) to amplify and obtain the hspX gene specifically expressed during the latent infection period of Mycobacterium tuberculosis (primers used: H1 / H2, the sequence is shown in ...

Embodiment 3

[0073] The identification of the tuberculosis vaccine of the present invention specifically includes the following aspects:

[0074] 1. Expression identification: LV-sp146, LV-hspX, LV-HIL47, and LV-HII1 were used to infect macrophage RAW264.7 cells, and cells not infected with lentivirus were set as controls. The cells in each group were collected 72 hours after infection, and the total RNA and protein of the cells were extracted. The expression levels of hspX and lrg47 / irgm1 were detected by RT-PCR technology, and the expression levels of HSP X and LRG47 / IRGM1 proteins were detected by Western blotting. The results showed that , after LV-hspX, LV-HIL47 and LV-HII1 infected RAW264.7 cells, they can all express the corresponding recombinant proteins effectively in the cells, such as Figure 1-Figure 4 shown. figure 1 RT-PCR was used to detect the mRNA transcription level of the recombinant gene in each lentivirus infection group and control group RAW264.7 cells; figure 2 RT...

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Abstract

The invention discloses tuberculosis vaccine and a preparation technology thereof. The tuberculosis vaccine is prepared from recombinant lentivirus expression plasmid pLenti-spHIL47 / pLenti-spHII1 andlentivirus packaging the recombinant lentivirus expression plasmid. The tuberculosis vaccine can obviously improve mycobacterium tuberculosis antigen presenting capacity of macrophage, can enhance a vaccine function for stimulating a body to generate antigen specificity immune response, can effectively activate the macrophage infected by mycobacterium tuberculosis, improves autophagy level of themacrophage and improves mycobacterium tuberculosis killing and cleaning functions of the macrophage. The tuberculosis vaccine disclosed by the invention can effectively promote the body to clean infected mycobacterium tuberculosis in the body; when the tuberculosis vaccine disclosed by the invention and tuberculosis chemotherapy drugs are combined and used, a tuberculosis treating period is remarkably shortened; when the tuberculosis vaccine disclosed by the invention and tuberculosis chemotherapy drugs are combined and used, tuberculosis relapsing can be remarkably delayed after the tuberculosis is treated; compared with other lentiviral vector vaccines, the tuberculosis vaccine disclosed by the invention has good macrophage targeting and use safety.

Description

technical field [0001] The invention belongs to the field of genetic engineering and the technical field of novel tuberculosis vaccines, and in particular relates to a tuberculosis vaccine and a preparation process thereof. Background technique [0002] Tuberculosis (TB) is a global health problem, generating 8 million new cases and 2 million deaths each year. The emergence of multi-drug and pan-drug resistant TB strains has only exacerbated this problem. The life cycle of Mtb has 3 phases. In the acute phase following initial infection, bacteria replicate in the host and express early secreted proteins that induce a host immune response. As the immune response begins to control the infection, Mtb enters a latent asymptomatic state in which the bacteria become non-replicating, parasitizing primarily within macrophages and encased in granulomas. The bacteria can persist in this latent state for many years in infected individuals, making diagnosis and treatment of the disea...

Claims

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Application Information

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IPC IPC(8): A61K39/04A61P31/06C12N15/867
Inventor 李俊明黄自坤江红罗清呙阳
Owner NANCHANG UNIV
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