Application of autovaccine preparation containing TGF beta 1

A technology of autologous vaccines and preparations, applied in the fields of immunology and animal vaccine manufacturing, which can solve the problems of incomplete inhibition of signaling pathways

Inactive Publication Date: 2010-07-21
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The possible reason is that the inhibitory effect of these inhibitors on the signaling pathway of TGF is incomplete

Method used

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  • Application of autovaccine preparation containing TGF beta 1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: The preparation of an autologous vaccine preparation fused with the full amino acid sequence of TGF-β1 and LTB carrier protein, comprising the following steps:

[0039] 1.1 Construction of recombinant plasmid pET-TGF-LTB plasmid

[0040] 1.1.1 Download the amino acid sequences of TGFβ1 and LTB respectively from the genebank website. The amino acid sequence of TGFβ1 is shown in SEQ ID NO: 1, and the amino acid sequence of LTB is shown in SEQ ID NO: 2. In order to prevent the B cell epitope of TGFβ1 from being affected by LTB, A flexible linker sequence (GGGGSGGGGSGGGGS) is added between the LTB and TGFβ1 dinucleotide sequences. The sequence is shown in SEQ ID NO:3. Wherein LTB is located at the 5' end of the flexible linker sequence, and TGFβ1 is located at the 3' end of the flexible linker sequence. The sequence is LTB-GGGGSGGGGSGGGGS-TGFβ1. According to the preferred codon conversion of Escherichia coli into a nucleotide sequence, see SEQ ID NO: 4 for t...

Embodiment 2

[0051] Example 2: Preparation of an autologous vaccine preparation expressed in fusion with the universal helper T cell epitope (PADRE), tetanus toxin T cell epitope peptide and TGF-β1 whole protein sequence:

[0052] The amino acid sequence of the universal helper T cell epitope (PADRE) is shown in SEQ ID NO: 5, the amino acid sequence of the tetanus toxin T cell epitope peptide (TTE) is shown in SEQ ID NO: 6, and the amino acid sequence of the constructed fusion protein is: PADRE- TTE-GGGGSGGGGSGGGGS-TGFβ1, the amino acid sequence is optimized according to the codons of E. coli, and the nucleotide sequence of the fusion protein is obtained by the method of whole gene synthesis, and cloned into the expression vector, and the expression strain is transformed to obtain the fusion protein. The specific preparation scheme is carried out with reference to Example 1 and "Molecular Cloning Experiment Guide".

Embodiment 3

[0053] Example 3: Effect of TGFβ1 autovaccine preparation on the number of CD4+CD25+ regulatory T cells in mice.

[0054] The prepared LTB-TGF or PARDE-TGF fusion protein was used to immunize mice for the second time, in which the fusion protein was immunized with complete Freund's adjuvant for the first time, and the fusion protein was immunized with incomplete Freund's adjuvant for the second time. mouse. The time interval between the second immunization was 15 days, and CD4 was measured 15 days after the second immunization + CD25 + The number and distribution of regulatory T cells. The reagents used were FITC rat anti-mouse CD4 monoclonal antibody and PE rat anti-mouse CD25 monoclonal antibody from eBioscience, USA, and CD4 was measured by BD FACSCaliburTM flow cytometer. + CD25 + The number and distribution of regulatory T cells. The results showed that autologous vaccine formulations were able to significantly reduce the number of regulatory T cells.

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Abstract

The invention discloses application of autovaccine preparation containing TGF beta 1. Adjuvant is added in the autovaccine preparation to prepare medicinal preparation for treating continuous pathogen infection of livestock. The vaccine preparation is fusion protein obtained by the way that part or total of the TGF beta 1 or part or total of mutant or total of similar gene sequences is recombined with at least one helper T cell capable of improving immunogenicity or the gene sequence of carrier protein to construct eukaryon or pronucleus expression plasmid and transform host cell for expression, or is directly prepared by eukaryon expression plasmid obtained by construction. The vaccine preparation can be directly used for preparing medicinal preparation for treating the continuous pathogen infection of livestock, induce domestic animals to generate antibody capable of neutralizing self TGF beta 1, and remove immunodepression function caused by too high level of TGF beta 1 in continuous process of infection; simultaneously, the vaccine preparation can depress the generation of inducible regulatory T cell at the persistent infection part of an organism, thus being beneficial to breaking the immune tolerance state of in-vivo pathogen by the organism and promoting the removing function of the organism on the continuously infectious pathogen.

Description

technical field [0001] The invention belongs to the technical field of immunology and animal vaccine production, and in particular relates to the application of TGFβ1 autovaccine preparation for treating persistent pathogenic infection of livestock. Background technique [0002] The phenomenon of persistent pathogenic infection of livestock is very common in modern farming industry. Although infected livestock often do not show obvious clinical symptoms, they have the ability to continuously shed toxins to the outside world; in female animals, they can also be passed on to the next generation through fetuses, and these persistent pathogens may also mutate in livestock and cause new infections. become an important source of infection. Persistent infection may also cause immune tolerance or paralysis, seriously affecting the effect of revaccination. This has brought great difficulties to the prevention and control and purification of infectious diseases in the aquaculture in...

Claims

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Application Information

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IPC IPC(8): A61K39/00A61K48/00A61P31/00A61P33/00
Inventor 许道军薛立群
Owner HUNAN AGRICULTURAL UNIV
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