Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application

A technology for recombining Saccharomyces cerevisiae and hepatitis B virus is applied in the field of preparation of recombinant Saccharomyces cerevisiae strains, which can solve the problems of limited treatment time and low curative effect, and achieve the effects of low cost, large-scale production, and good application and development prospects.

Active Publication Date: 2021-12-21
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] IFN-α2, as the most studied subtype, has been used in the treatment of chronic hepatitis B virus (HBV) infection with limited duration of treatment and sustained virological response advantage, but its efficacy is still relatively low

Method used

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  • Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application
  • Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application
  • Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Construction of GPD-HBVs-TU and GPD-IFNα14-TU vectors

[0036] (1) Amplification of HBVs and IFNα14 genes

[0037] According to the sequence structure of HBVs and IFNα14 gene ( figure 1 , 2 shown), design and synthesize primers, TU-HBVs-F (SEQ ID NO.7: gacgataagg taccaggatc catgggagga tggtcatcaa agc) and TU-HBVs-R (SEQ ID NO.8: tgctggatat ctactggatc cgagtatacg tgtcaggagg aagaatc), TU-IFNα14-F (SEQ ID NO. 9: gatgtgcttc gattccccgg gtgtaacctt tcacaaacac attcact) and TU-IFNα14-R (SEQ ID NO. 10: atctgtaagt ctagacccgg ggtcctttct tctcagccgc tt). According to the reported HBVs gene sequence (Genbank number AF384371.1) and IFNα14 gene sequence (Genbank number NM_002172.3), the genes of HBVs protein and IFNα14 protein were optimized and artificially synthesized, and pET28a(+)-HBVs plasmid was constructed and pET28a(+)-IFNα14, using the plasmid as a template to amplify the coding genes of HBVs (1-200) and IFNα14 (24-189). The PCR amplification system is:

[0038] ...

Embodiment 2

[0045] Example 2. Construction and detection of recombinant Saccharomyces cerevisiae strains expressing IFNa14 protein and human hepatitis B virus S protein simultaneously

[0046] (1) Construction of Yeast Transformation Fragment

[0047] The vector GPD-HBVs-TU was cut with BsaI, and at the same time, the homology arm plasmids (URR1 and URR2) and the selectable marker plasmid (LEU) were cut with BsmB I. Refer to the experimental steps of Dai's research group [6] , URRs homology arms, LEU selective tags, and GPD-HBVs-TU transcription units were spliced ​​according to the specific prefix and suffix sequences, T4 ligase was ligated overnight at 16°C, and the spliced ​​products were used for yeast transformation.

[0048] The vector GPD-IFNα14-TU was cut with BsaI, and at the same time, the homology arm plasmids (SUR1 and SUR2) and the selectable marker plasmid (Trp) were cut with BsmB I. Refer to the experimental steps of Dai's research group [6] , the SURs homology arm, Trp ...

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Abstract

The invention discloses a recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as a preparation method and application. The recombinant saccharomyces cerevisiae strain contains 1-200 amino acids of HBVs protein and 24-190 amino acids of IFN alpha 14. Two phenotypes are integrated into one yeast strain, so that the recombinant yeast strain JDY52-HBVs-IFN alpha 14 capable of simultaneously displaying the HBVs protein and secreting the IFN alpha 14 protein on the surface is obtained. The recombinant saccharomyces cerevisiae strain is used for preparing a hepatitis B virus oral vaccine, the protective immune response of an organism is stimulated through an oral administration way, and more effective immune protection is exerted by virtue of the regulating effect of yeast cell wall polysaccharide on the congenital immune system of the organism. Compared with the existing vaccine, the oral recombinant yeast preparation is low in cost, can realize large-scale enlarged production, is safe and reliable, has good application and development prospects, and provides a choice for immunization and treatment of hepatitis B virus.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a preparation method and application of a recombinant Saccharomyces cerevisiae strain expressing IFNa14 protein and human hepatitis B virus S protein. Background technique [0002] Viral hepatitis B is a worldwide epidemic disease caused by hepatitis B virus (HBV) infection, especially in developing countries, the morbidity and mortality have been high [1] . HBV is only 3200bp, which is a fairly small virus. There are four ORFs in its genome, encoding the following proteins: Core protein and pre-core protein, Pol protein, X protein, and S protein (L, M, S). Core is a nucleocapsid protein; Pre-core does not know what function it has now, it is not necessary for virus replication, but it may be related to the suppression of the host's immune response; X protein is important for virus replication, and is also related to the occurrence of liver cancer Related; S prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/62C12N15/81A61K39/29A61K38/21A61K48/00A61P31/20C12R1/865
CPCC07K14/56C07K14/005C12N15/81A61K39/12A61K38/212A61K48/0008A61K48/005A61K48/0075A61P31/20C12N2730/10122C12N2730/10134C07K2319/00A61K2039/53A61K2039/542Y02A50/30
Inventor 黄金海孙瑞骐郭艳余
Owner TIANJIN UNIV
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