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A tandem polypeptide and its application in immune protection against Mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and Mycobacterium tuberculosis, applied in the field of immunology, can solve the problems of limited immune protection effect, need of adjuvant help, multiple vaccinations, etc., and achieve the effect of important application value

Active Publication Date: 2021-04-13
中国人民解放军总医院第八医学中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the deepening of research, people found that subunit vaccines also have shortcomings, such as the need for multiple vaccinations, the need for adjuvant help, and limited immune protection effects.

Method used

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  • A tandem polypeptide and its application in immune protection against Mycobacterium tuberculosis
  • A tandem polypeptide and its application in immune protection against Mycobacterium tuberculosis
  • A tandem polypeptide and its application in immune protection against Mycobacterium tuberculosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, artificial synthesis Mycobacterium tuberculosis Th1 epitope peptide

[0070] 1. Using bioinformatics to predict Mycobacterium tuberculosis Th1 dominant epitopes

[0071] 1. Obtain the amino acid sequences of target proteins such as Mycobacterium tuberculosis Ag85A, Ag85B, CFP21, and PPE18 from the NCBI database, and then use the IEDB MHC-Ⅱ database (URL: http: / / tools.iedb.org / mhcii / ) to predict HLA-DRB1*01:01 restricted epitope.

[0072] 2. Use IEDB recommended (URL: http: / / www.iedb.org / ), Consensusmethod, Combinatorial library, NN-align (netMHCII-2.2), SMM-align (netMHCII-1.1), Sturniolo and NetMHCIIpan Methods The HLA-DRB1*01:01 restricted epitopes predicted in step 1 were predicted, and then scored by comprehensive ranking. Epitopes with a score of less than 10 in the comprehensive ranking were Th1 dominant epitopes, and the lower the score, the higher the affinity.

[0073] Consensus method is described in the following documents: Wang P, Sidney J, K...

Embodiment 2

[0085] Example 2, using four independent ELISOPT experiments to screen positive epitope peptides

[0086] 1. The first ELISPOT experiment (repeated 2 times)

[0087] A. Epitope peptide stimulation group

[0088] 1. Preparation of splenocyte suspension

[0089] (1) 100 μL of inactivated Mycobacterium tuberculosis bacteria liquid (about 5×10 6 PFU) and 100 μL complete Freund's adjuvant were mixed, and then C57BL / 6 mice were immunized.

[0090] A total of 10 mice were immunized.

[0091] (2) The mice were killed on the 15th day after immunization, and then placed in 75% (v / v) ethanol water solution, taken out after 10 minutes, and the spleen was dissected.

[0092] (3) Take a sterile petri dish, add 10mL of 1640 cell culture medium, then place a sterile 200-mesh copper mesh in the sterile petri dish, then place the spleen on the copper mesh, and push the head of the rod with a syringe (without Bacteria) Squeeze gently to disperse the splenocytes.

[0093] (4) Prepare the EL...

Embodiment 3

[0176] Embodiment 3, protective evaluation of tandem polypeptide ACP

[0177] 1. Immunization of mice

[0178] Female humanized mice or female wild-type mice aged 6-7 weeks and weighing 16-18 g were randomly divided into experimental group (i.e. ACP group), negative control group (i.e. PBS group), and positive control group (i.e. PBS group). BCG group) and strengthening group (BCG+ACP group) in 4 groups, with 6 or 7 rats in each group. Each group was treated as follows:

[0179] Experimental group: on the 28th day of adaptive feeding, subcutaneously immunized once with 30 μg tandem polypeptide ACP prepared in 200 μL CpG adjuvant, and then intraperitoneally immunized twice with 20 μg tandem polypeptide ACP prepared in 200 μL CpG adjuvant, with an interval of 14 days between each immunization;

[0180] Negative control group: on the 28th day of adaptive feeding, subcutaneously immunize once with 200 μL mixed solution (mixed with 30 μg CpG adjuvant freeze-dried powder and 200 μ...

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Abstract

The invention discloses a tandem polypeptide and its application in immune protection against Mycobacterium tuberculosis. The tandem polypeptide contains polypeptide A with amino acid sequence as shown in SEQ ID NO:2, polypeptide B with amino acid sequence as shown in SEQ ID NO:3 and polypeptide C with amino acid sequence as shown in SEQ ID NO:4. Experiments have shown that booster immunization with tandem peptides after BCG primary immunization can induce a significant reduction in the number of Mycobacterium tuberculosis colonies in the liver and lung of humanized C57BL / 6 mice, and its immune protection efficiency is better than that of BCG alone immunization group; There was no significant difference in the number of Mycobacterium tuberculosis colonies in liver and lung of C57BL / 6 mice. It can be seen that the tandem polypeptide can inhibit the proliferation of mycobacterium tuberculosis in humanized animals; accordingly, a vaccine for mycobacterium tuberculosis can be prepared. The invention has important application value.

Description

technical field [0001] The invention belongs to the field of immunology, and specifically relates to a tandem polypeptide and its application in immune protection against mycobacterium tuberculosis. Background technique [0002] Vaccination is the best way to eliminate tuberculosis (TB) infection, but there is still a lack of effective vaccines. The earliest vaccine against tuberculosis was BCG vaccine, but a large number of studies have shown that although BCG has a preventive effect on children with severe tuberculosis (such as miliary tuberculosis and tuberculous meningitis), its protective effect decreases with age, and BCG has a protective effect on People who have been infected with tuberculosis are basically ineffective. Compared with the BCG vaccine, the subunit vaccine uses only certain protein antigens that can induce the body to produce a protective immune response instead of whole bacterial antigens, and its side effects after immunization are significantly redu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K39/04A61P31/06
CPCA61K39/04A61P31/06C07K14/35C07K2319/40
Inventor 龚文平吴雪琼梁艳王兰王杰薛勇米洁
Owner 中国人民解放军总医院第八医学中心