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Truncated expression of abrin toxin a chain protein antigen and its application

A kind of abrin toxin and protein technology, applied in the field of abrin toxin A chain protein antigen, has achieved significant application value and far-reaching application prospects

Active Publication Date: 2016-06-08
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chinese scholars have made initial progress in the detection and detection of abrin toxin biological warfare agents, but the research on its vaccine as a medical protection has just started. There is no effective immunotherapy and protection against potential abrin toxin terrorist attacks. Therefore, it is urgent to establish and develop protective equipment with independent intellectual property rights, to ensure timely adoption of emergency measures and countermeasures, and to minimize the damage caused by bioterrorism attacks

Method used

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  • Truncated expression of abrin toxin a chain protein antigen and its application
  • Truncated expression of abrin toxin a chain protein antigen and its application
  • Truncated expression of abrin toxin a chain protein antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the preparation of tATA4 fragment

[0025] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing (coding the tATA4 fragment shown in sequence 2 of the sequence listing).

[0026] 2. Using the double-stranded DNA molecule synthesized in step 1 as a template, perform PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0027] F1: 5'-CCG GAATTC GAAGATCGCCCGATCA-3';

[0028] R1: 5'-CTA GCTAGC ATCCGGCTGAAATGCCGTA-3'.

[0029] 3. Digest the PCR amplified product in step 2 with restriction endonucleases EcoRI and NheI, and recover the digested product.

[0030] 4. Digest the pET-His expression vector with restriction endonucleases EcoRI and NheI, and recover the vector backbone of about 2.9 kb.

[0031] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain a recombinant plasmid. According to the sequencing results, the structure of the recom...

Embodiment 2

[0040] Embodiment 2, preparation full-length protein

[0041] 1. Between the EcoRI and NheI restriction sites of the pET-His expression vector, the double-stranded DNA molecule shown in sequence 5 of the sequence listing is inserted forward (the double-stranded DNA molecule shown in sequence 5 of the sequence listing encodes A chain protein shown in sequence 6) to obtain a recombinant plasmid.

[0042] 2. Introduce the recombinant plasmid obtained in step 1 into Escherichia coli BL21(DE3) to obtain recombinant bacteria.

[0043] 3. Inoculate the recombinant bacteria obtained in step 2 into the liquid LB medium, shake and cultivate to OD at 37°C and 200rpm 600nm About 0.6; then, add IPTG to make the concentration 0.01mM, then shake culture at 20°C and 200rpm for about 12-14h, and then centrifuge at 8000g for 10min to collect the bacteria.

[0044] 4. Take the bacterial cells obtained in step 3, resuspend them with protein purification solution A (solvent pH 7.5, 20mM Tris-cl ...

Embodiment 3

[0048] Embodiment 3, the performance analysis of tATA4 fragment

[0049] 1. Western blot analysis

[0050] The tATA4 fragment solution prepared in Example 1 was subjected to Western blot analysis (the primary antibody used in Western blot analysis was the serum collected after immunizing rabbits with the A chain protein solution prepared in Example 2, and the secondary antibody was goat anti-rabbit IgG), showing There is a specific color band around 22KDa, indicating that the tATA4 fragment can be recognized by the A chain protein-specific serum antibody and has good antigenicity.

[0051] 2. Toxicity analysis

[0052] 1. Determination of the killing effect of tATA4 fragment and A chain protein on BEAS-2B cells by MTS method

[0053] (1) Seed BEAS-2B cells into a 96-well plate (10,000 cells / well), and culture overnight to allow the cells to adhere to the wall.

[0054] (2) After completing step (1), divide the wells into three groups (add the 3-fold gradient dilution of the...

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Abstract

The invention discloses a truncated-expressed protein antigen of a chain A of abrin and application thereof. The invention provides a tATA4 segment which is (a) or (b) or (c) or (d): (a) protein comprising an amino acid sequence shown by a sequence 2 in a sequence table, (b) protein having the amino acid sequence shown by the sequence 2 in the sequence table, (c) protein comprising the amino acid sequence shown by a sequence 4 in the sequence table, and (d) protein which is obtained through substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of the sequence 2 or the sequence 4, has the same function and is derived from the sequence 2 or the sequence 4. The tATA4 segment provided by the invention can be used as the active component of vaccine resisting abrin and has important application value and profound application prospect.

Description

technical field [0001] The present invention relates to truncated expression abrin toxin A chain protein antigen and application thereof. Background technique [0002] With the rapid development of biotechnology and the development and utilization of biotoxins, the importance of toxins in weapons of mass destruction has been highlighted, and the threat of biotoxins and their bioterrorism is increasing day by day. As a Class B biological warfare agent, abrin toxin is included in the international biological warfare agent checklist. It is highly similar to ricin in molecular structure and mechanism of action, and is composed of A-B chain structure (A chain is the effect chain, with RNAN-glycosidase activity; the B chain is the binding chain, which mediates the A chain into the cell to play a toxic role). Abbrin toxin mouse intraperitoneal LD 50 It is 0.04μg / kg, and its toxicity is 75 times that of ricin. Acacia beans are mainly distributed in subtropical regions and are wid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/70C12N1/21A61K39/00A61P39/02C12R1/19
CPCA61K39/00C07K14/415
Inventor 王景林张弢康琳高姗
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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