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Recombination recin toxin B chain truncated protein and expressing method and application thereof

A technology for ricin and truncated proteins, which is applied in the field of bioengineering, can solve the problems of poor refolding protein stability and low refolding rate of recombinant ricin B chain protein, achieve good immunogenicity, reduce vaccine dosage, The effect of enhanced stability

Active Publication Date: 2016-04-13
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve technical problems such as low renaturation rate of existing recombinant ricin B chain protein and poor stability of refolded protein, the present invention provides a recombinant ricin B chain truncated protein and its expression method and application

Method used

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  • Recombination recin toxin B chain truncated protein and expressing method and application thereof
  • Recombination recin toxin B chain truncated protein and expressing method and application thereof
  • Recombination recin toxin B chain truncated protein and expressing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Escherichia coli expression system expresses recombinant ricin B chain truncated protein

[0027] 1. The acquisition of Escherichia coli expression engineering bacteria BL21(DE3) / PET28a-RTB

[0028] Under the condition that the amino acid sequence remains unchanged, codon optimization is carried out according to the existing ricin B chain (rRTB) sequence, specifically: there are 9 cysteines in the ricin B chain protein gene, and its amino acid The first five amino acids in the sequence were truncated to obtain the ricin B chain truncated gene sequence, and the codon-optimized full sequence of the ricin B chain was artificially synthesized, and its nucleotide sequence was sequence 1 in the sequence list, Its amino acid sequence is sequence 2 in the sequence list.

[0029] According to the above-mentioned ricin B chain codon-optimized full sequence as a template, use rRTBsenseprimer (sequence 3) and rRTBanti-senseprimer (sequence 4) for PCR amplification. The P...

Embodiment 2

[0038] Example 2 Recombinant ricin B chain truncated protein as a vaccine adjuvant to enhance the immune effect test of rabies vaccine

[0039] 1. Experimental animals and grouping

[0040] 50 7-week-old Kunming mice were randomly divided into 5 groups, 10 in each group, no adjuvant virus control group, aluminum hydroxide adjuvant vaccine group, rabies vaccine+20ug recombinant ricin B chain truncated protein ( RTB) vaccine group, rabies vaccine+40ug recombinant ricin B chain truncated protein (RTB) vaccine group, rabies vaccine+80ug recombinant ricin B chain truncated protein (RTB) vaccine group.

[0041] 2. Experimental steps

[0042] The culture titer is 10 6 TCID 50 Rabies virus JX08-45CC strain (120th generation) per mL was inactivated by β-propiolactone, and the unadjuvanted virus control group, aluminum hydroxide adjuvant vaccine group, rabies vaccine+recombinant ricin B chain In the truncated protein vaccine group, the mice were mixed with the virus solution and imm...

Embodiment 3

[0045] Example 3 Recombinant ricin B chain truncated protein is used as a vaccine adjuvant to enhance the immune effect test of influenza virus vaccine

[0046] 1. Experimental animals and grouping

[0047] Thirty 7-week-old Kunming mice were randomly divided into 3 groups, 10 in each group, normal control group (PBS), H1N1VLP vaccine group, H1N1VLP+40ug recombinant ricin B-chain truncated protein (RTB) vaccine group.

[0048] 2. Experimental steps

[0049] After immunization at week 0 and week 3, 50 μL / rat was injected intramuscularly into the hind limbs, and the model was established with influenza virus FM1-6-E2 nasal drop at the fifth week, and the challenge dose was 5LD50, 50 μL / rat.

[0050] 3. Results

[0051] 1) Survival rates are shown in Table 1. From 1 to 4 weeks, the survival rates of the three groups were all 100%. From 5 to 14 weeks, the H1N1VLP vaccine group and the H1N1VLP+40ug recombinant ricin B chain truncated protein vaccine group were immune The surviva...

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Abstract

The invention discloses a recombination recin toxin B chain truncated protein and an expressing method and application thereof, and relates to the field of bioengineering. The technical problems that an existing recombination recin toxin B chain protein is low in renaturation, poor in renaturation protein stability and the like are solved. The nucleotide sequence of the recombination recin toxin B chain truncated protein is shown in the SEQ ID NO:1 in a sequence table. The amino acid sequence of the recombination recin toxin B chain truncated protein is shown in the SEQ ID NO:2 in the sequence table. The expressing method of the recombination recin toxin B chain truncated protein comprises the step of expressing the recombination recin toxin B chain truncated protein through an escherichia coli expression system. According to the recombination recin toxin B chain truncated protein, the renaturation rate is improved, the stability is enhanced, the immunogenicity is good, the vaccine immunity effect is enhanced, the vaccine consumption is lowered, and the recombination recin toxin B chain truncated protein can be used as adjuvants of various vaccines such as inactivated vaccines, activity reduction vaccines, split vaccines, DNA vaccines, recombinant vaccines, subunit vaccines and polypeptide protein vaccines.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant ricin B chain truncated protein and its expression method and application. Background technique [0002] Ricin is a heterodimeric glycoprotein consisting of chain A (RTA) and chain B (RTB), which are connected by disulfide bonds. RTA is a ribosome-inactivating protein that inhibits protein synthesis in mammalian cells. RTB is non-toxic, has lectin activity, and assists A chain to enter cells to perform biological functions. RTB is a two-leaf-shaped molecule composed of two identically folded topological globular domains, each of which includes three subdomains (α, β, γ), and only 1α and 2β have significant galactose binding Active, RTB can bind different sugar structures and mediate a variety of biological processes, including cell-host, pathogen interactions, and innate immune responses. Contents of the invention [0003] In order to solve technical pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/70A61K39/39A61K39/145A61K39/205A61P31/14A61P31/16
CPCA61K39/12A61K39/39A61K2039/55516C07K14/415C12N2760/16034C12N2760/16134
Inventor 刘文森许娜孙成彪黄艳霞陈华鑫
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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