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Preparing method and application of cell immunologic adjuvant TSA-41

A technology of TSA-41 and cellular immunity, applied in the field of biomedicine, can solve problems such as nucleic acid safety hazards, and achieve the effects of being beneficial to industrial production, improving preparation efficiency, and avoiding process steps

Active Publication Date: 2016-12-21
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that aluminum hydroxide cannot produce an effective adjuvant function for this type of vaccine, and adjuvants that have been proven to be effective in improving cellular immunity in laboratory studies have non-methylated cytosine-guanine nucleotide motifs Oligodeoxynucleotide (CpG ODN), etc., but as a nucleic acid, CpG ODN still has great hidden dangers in its safety, so it has not been approved for marketing for a long time

Method used

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  • Preparing method and application of cell immunologic adjuvant TSA-41
  • Preparing method and application of cell immunologic adjuvant TSA-41
  • Preparing method and application of cell immunologic adjuvant TSA-41

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 TSA-41 adjuvant

[0039] (1) Synthesize the gene sequence of SEQ ID NO.1 by artificial gene synthesis technology, and insert the gene of SEQ ID NO.1 into pET28a Escherichia coli expression plasmid through the two restriction sites of NdeI and XbaI to obtain the expression of the gene of SEQ ID NO.1 The sequence of recombinant plasmid SEQ ID NO.1-pET28a.

[0040](2) Take a 1.5 ml centrifuge tube containing 50 microliters of BL21 (DE3) Escherichia coli competent cells and place it on ice for 5 minutes, add 50 nanograms of SEQ ID NO.1-pET28a recombinant plasmid, mix gently, And incubate on ice for 30 minutes; place the centrifuge tube in a 42°C water bath for 90 seconds, quickly take it out and place it on ice for 5 minutes; add 900 microliters of LB liquid medium to the centrifuge tube, and culture with shaking at 37°C for 45 Minutes, take 200 microliters of the culture product and spread it on an LB solid medium plate with a diameter of 1...

Embodiment 2

[0054] Example 2 Effect of TSA-41 adjuvant on proliferation of mouse lymphocytes in vitro

[0055] (1) The C57 / BL mice were sacrificed, and the spleen was taken and crushed gently under sterile conditions to obtain a spleen cell suspension.

[0056] (2) The obtained spleen cell suspension was treated with erythrocyte lysate for 5 minutes, centrifuged at 1000 rpm to discard the supernatant, then washed twice with serum-free RPMI-1640 medium, and finally the cell pellet was resuspended in a certain amount of serum RPMI In -1640 medium, the mouse spleen lymphocyte suspension was obtained.

[0057] (3) Dilute the mouse spleen lymphocyte suspension to 5×10 6 cells / ml, seeded in 96-well cell culture plate, 100 μl per well.

[0058] (4) Dilute the TSA-41 adjuvant with cell culture medium to a series concentration of 4 mg / ml, 2 mg / ml, 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, 0.125 mg / ml, 0.063 mg / ml ml, 0.032 mg / ml, and 0.016 mg / ml, take 1 microliter of adjuvant of each concentration and ad...

Embodiment 3

[0062] Example 3 Enzyme-linked dot immunoassay detection of TSA-41 adjuvant's immune enhancement effect on human papillomavirus recombinant protein vaccine for treatment

[0063] (1) There were three C57 / BL mice in each group of experimental animals, which were divided into three groups: TSA-41 adjuvant group, human papillomavirus recombinant protein vaccine group for treatment, human papillomavirus recombinant protein vaccine for treatment and TSA- 41 adjuvant co-immunization groups. Among them, the inoculation volume of human papillomavirus recombinant protein vaccine for treatment was 100 micrograms, and the inoculation volume of TSA-41 adjuvant was 5 micrograms. Each mouse was boosted immunized two days apart, and inoculated three times in total.

[0064] (2) The mice were sacrificed on the 10th day of the initial immunization, and the spleen was removed and crushed gently under aseptic conditions to obtain a spleen cell suspension.

[0065] (3) The obtained spleen cell s...

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Abstract

The invention relates to the technical field of biological medicine, in particular to a preparing method and application of a cell immunologic adjuvant TSA-41 used for a vaccine adjuvant. According to the preparing method, a gene sequence SEQ ID NO.1 is artificially designed, the SEQ ID NO.1 recombination gene sequence is inserted into an escherichia coli expression system through the gene engineering technology, the soluble recombinant protein TSA-41 with a dipolymer structure is efficiently expressed through escherichia coli, obtained TSA-41 recombinant protein is separated and purified, and finally high-purity and high-activity recombinant protein is obtained. The recombinant protein as the adjuvant can effectively improve the immune effect of cell immune type biological vaccines, the technological step of recombinant protein refolding is omitted, the preparing efficiency is greatly improved, and industrial production is promoted.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a preparation method and application of a cellular immune adjuvant TSA-41 used as a vaccine adjuvant. Background technique [0002] Biological products such as vaccines exert their effects by stimulating the body's immune system and producing immune substances (such as antibodies), and humoral immunity, cellular immunity or cell-mediated immunity appear in the human body. It is a product brought about by the deepening of human understanding of things and the progress of science and technology. It is a great weapon for human beings to resist the threat of external adverse factors and improve the quality of health. However, the active ingredients of a single vaccine are often affected by various biological pathways during the process of immune presentation, making it impossible to achieve the expected immune effect. Therefore, some methods that can enhance the body's immune res...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/00C07K1/22A61K39/39A61K39/12A61P31/20
CPCA61K39/12A61K39/39A61K2039/55516C07K14/00C12N15/70C12N2710/20034C12N2800/101
Inventor 高孟高丽美朱赟罗永能吴洁陈刚庄昉成毛子安毛江森
Owner ZHEJIANG PUKANG BIOTECH
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