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Recombinant ricin B-chain truncated protein and its expression method and application

A technology of ricin and truncated protein, applied in the field of bioengineering, can solve the problems of low renaturation rate of recombinant ricin B-chain protein, poor stability of renatured protein, etc., and achieves reduction of vaccine dosage, good immunogenicity, The effect of increasing the renaturation rate of B chain

Active Publication Date: 2019-06-11
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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  • Recombinant ricin B-chain truncated protein and its expression method and application
  • Recombinant ricin B-chain truncated protein and its expression method and application
  • Recombinant ricin B-chain truncated protein and its expression method and application

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 rRTB sense primer (sequence 3) and rRTB anti-sense primer (sequence 4) for PCR amplification, 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 groups

[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 immun...

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 groups

[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 survival ...

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Abstract

The recombinant ricin B-chain truncated protein and its expression method and application relate to the field of bioengineering and solve the technical problems of the existing recombinant ricin B-chain protein such as low renaturation rate and poor renaturation protein stability. The nucleotide sequence of the recombinant ricin B-chain truncated protein is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing. The invention provides a method for expressing the recombinant ricin B chain truncated protein, which expresses the recombinant ricin B chain truncated protein through an Escherichia coli expression system. The recombinant ricin B chain renaturation rate of the present invention is improved, the stability is enhanced, the immunogenicity is good, the immune effect of the vaccine is enhanced, the dosage of the vaccine is reduced, and it can be used as an inactivated vaccine, an attenuated vaccine, a split vaccine, a DNA vaccine, or a recombinant vaccine , subunit vaccine, polypeptide protein vaccine and other vaccine adjuvants.

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 Patents(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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