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Bovine infectious rhinotracheitis virus multi-epitope recombinant chimeric protein and application thereof

A rhinotracheitis virus and chimeric protein technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as potential safety hazards, imperfect gene deletion technology, and strong virulence

Active Publication Date: 2019-07-12
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the inactivated vaccine can induce better humoral immunity, the immunity period is short, and it cannot be differentiated from natural infection, so it brings certain obstacles to the eradication of IBR.
The attenuated vaccine can induce good humoral immunity and cellular immunity, and the immunity period is long, but there is a possibility of strong virulence, and there are certain safety hazards
Gene-deleted seedlings have been used as the main means to prevent and purify IBR in some developed countries in Europe, but the gene-deleted technology is not perfect, and there are still certain risks

Method used

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  • Bovine infectious rhinotracheitis virus multi-epitope recombinant chimeric protein and application thereof
  • Bovine infectious rhinotracheitis virus multi-epitope recombinant chimeric protein and application thereof
  • Bovine infectious rhinotracheitis virus multi-epitope recombinant chimeric protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The design of embodiment 1 bovine infectious rhinotracheitis virus multi-epitope tandem gene

[0041] See Table 1 for the epitope information of the bovine infectious rhinotracheitis virus used in this example. Use AAYAAYLinker to arrange and combine tetanus toxin universal T cell epitope P2 and gB, gC and gD epitopes in different ways, analyze their antigenicity with DNAStar Protean software, select a combination with better antigenicity parameters, and the connection sequence is as follows: figure 1 As shown, and set restriction sites on the tandem genes. The designed tandem gene sequence and bovine IL-6 gene sequence (with Sal I and Hind III restriction sites introduced at both ends) were entrusted to Beijing Huada Gene Company for synthesis.

[0042] Table 1 Sequence information of P2, bovine IL-6 and gB / gC / gD epitope

[0043]

Embodiment 2

[0044] Embodiment 2 Contains the construction of the recombinant expression vector of fragment of interest

[0045] Transform the synthetic pUC-57-P2-gB / gC / gD plasmid containing the target fragment into DH5α competent cells, pick positive single colonies into LB liquid medium, and extract the plasmid after 12 hours of culture, the obtained plasmid and pET The -28a empty vector was double-digested with Nhe I and Hind III respectively. The double-digestion system is shown in Table 2, and the double-digestion results are shown in figure 2 . Use the OMEGA Gel Extraction Kit to quickly recover double-digested products. The recovered P2-gB / gC / gD target fragment was ligated to the pET-28a expression vector. The ligation system is shown in Table 3, thereby obtaining the pET-28a-P2-gB / gC / gD recombinant plasmid. For the results of double enzyme digestion identification of the recombinant plasmid, see image 3 , see Table 4 for the double enzyme digestion system. DNA sequencing was ...

Embodiment 3

[0075] Example 3 Expression of Bovine Infectious Rhinotracheitis Virus Multi-epitope Recombinant Protein

[0076] The identified correct pET-28a-P2-gB / gC / gD epitope-bovine IL-6 recombinant plasmid was transformed into E.coli BL21(DE3) competent cells by heat shock method. Pick a monoclonal colony and inoculate it in LB liquid medium containing 50 μg / mL kanamycin, when OD 600 When it reaches 0.5, add IPTG (final concentration is 1mM), induce culture at 37°C for 4h, collect the bacteria, resuspend the bacteria in PBS, put them on ice and ultrasonically break, collect the supernatant and precipitate, and carry out SDS-PAGE electrophoresis to identify the protein Expression situation, the electrophoresis of the SDS-PAGE of P2-gB / gC / gD epitope-bovine IL-6 recombinant protein is as follows Image 6 shown.

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Abstract

The invention provides a bovine infectious rhinotracheitis virus multi-epitope recombinant chimeric protein and an application thereof. The recombinant chimeric protein is formed by rigid linker series connection of a tetanus toxin general T cell epitope polypeptide P2, a bovine infectious rhinotracheitis virus gB antigen epitopes gB-A and gB-B, bovine infectious rhinotracheitis virus gC antigen epitopes gC-A and gC-B, bovine infectious rhinotracheitis virus gD antigen epitopes gD-A, gD-B and gD-C and bovine IL-6. The anti-bovine infectious rhinotracheitis virus antibody level induced by the bovine infectious rhinotracheitis virus multi-epitope recombinant chimeric protein provided by the invention is significantly higher than that of other control groups, and the recombinant chimeric protein is indicated to have good immunogenicity.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and in particular relates to multi-epitope recombinant chimeric protein of bovine infectious rhinotracheitis virus and its application. Background technique [0002] Infectious bovine rhinotracheitis virus (IBRV), also known as bovine herpesvirus 1 (BHV-1), is an important pathogen that endangers the development of the cattle industry and can cause severe respiratory diseases . After acute infection, BHV-1 can establish a lifelong latent infection in the trigeminal ganglion or dorsal ganglion. When stimulated by stress factors, BHV-1 in a state of latent infection can be activated and excreted again, giving control and elimination The associated diseases caused by BHV-1 pose great difficulties. [0003] Bovine infectious rhinotracheitis virus is an important pathogen that causes infectious diseases of cattle worldwide. It can cause symptoms such as high fever, dyspnea,...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/245A61P31/22C12R1/19
CPCC07K14/005C12N15/70A61K39/12A61P31/22C12N2710/16022C12N2710/16034A61K2039/552Y02A40/70
Inventor 冉旭华闻晓波仝晓丹范春玲张旭倪宏波毕莹
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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