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Avian influenza HA-Fd fusion protein, and preparation method and vaccine thereof

A fusion protein and avian influenza technology, applied in the field of biomedicine, can solve the problems of poor immune activity and achieve good immunogenicity, good protection, and high-efficiency and quality effects

Inactive Publication Date: 2020-09-15
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the conformation of the HA protein in the subunit vaccine in which HA is the main active ingredient is quite different from that of the natural HA protein, so the immune activity is poor and cannot effectively induce the body to produce an immune response

Method used

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  • Avian influenza HA-Fd fusion protein, and preparation method and vaccine thereof
  • Avian influenza HA-Fd fusion protein, and preparation method and vaccine thereof
  • Avian influenza HA-Fd fusion protein, and preparation method and vaccine thereof

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preparation example Construction

[0032] In some optional embodiments, the preparation method includes the following steps:

[0033] (a) Codon-optimizing the gene encoding the avian influenza HA-Fd fusion protein and synthesizing the optimized gene.

[0034] (b) Insert the artificially synthesized fusion protein gene into the eukaryotic expression vector pcDNA TM In 3.3-TOPO, positive recombinant vectors were screened out through enzyme digestion identification and sequencing, and the pcDNA3.3-HA-Fd plasmid was obtained.

[0035] (c) pcDNA3.3-HA-Fd plasmid, using FreeStyle TM MAX Reagent was transfected into CHO-S cells and named as CHO-HA-Fd. The avian influenza HA-Fd fusion protein was transiently expressed in CHO cells, and the cell supernatant containing the avian influenza HA-Fd fusion protein was obtained after purification, and stored at 4°C for future use.

[0036] pcDNA TM 3.3-TOPO is a bicistronic cloning vector, which can be applied to a variety of mammalian cell expression systems, using pcDNA ...

Embodiment 1

[0048] pcDNA TM 3.3-HA vector construction:

[0049] 1.1 Gene synthesis of avian influenza HA-Fd:

[0050] Obtain the avian influenza HA gene, GenBank accession number: ABW90137.1.

[0051] The HA gene was spliced ​​with the Fd sequence, and the codon-optimized nucleotide sequence was optimized as shown in SEQID NO.4, and then a His tag with 6 histidines was added to the N-terminal, and a His tag was added at both ends of the gene. The ends were inserted into restriction sites HindIII and BamHI, and sent to a synthetic company for gene synthesis.

[0052] 1.2 pcDNA TM 3.3-HA-Fd plasmid construction:

[0053] 1.2.1 Ligation reaction: Ligation of the recovered PCR product and pcDNA3.3, the ligation system (10 μL / tube) is as follows:

[0054] pcDNA3.3 vector 1μL Recovery of digested products 2μL wxya 2 o

2μL Solution I 5μL

[0055] Ligation procedure: connect overnight on the ligation instrument at 16°C.

[0056] 1.2.2 Transformatio...

Embodiment 2

[0063] Preparation of HA-Fd trimer fusion protein by CHO expression system:

[0064] 1.1 CHO-S cell culture: medium: Hycell+8mM GlutaMAX; medium purchased from HyClone; CHO-S cell culture conditions: 37°C, 5% CO 2 , 125rpm; cultivated to a cell density of 1.5×10 6 / ml, transfection when cell viability ≥ 90%, cell density and viability are counted and observed by trypan blue staining, and transfection reagent is FreeStyle TM MAX Reagents, FreeStyle TM MAX reagent was purchased from Invitrogen, and cultured in a shaking shaker incubator after transfection, culture parameters: 37°C, 5% CO 2 , 125rpm.

[0065] 1.2 Plasmid extraction: using PureLink TM Hipure Plasmid Maxiprep Kit (endotoxin-free) kit for large plasmid extraction, please refer to the kit manual for the extraction method.

[0066] 1.3 Transfection: Using FreeStyle TM MAX Reagent was used as a transfection reagent to convert pcDNA TM 3. The 3-HA-Fd plasmid was transfected into CHO-S cells, and after 14 days ...

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Abstract

The invention provides an avian influenza HA-Fd fusion protein as well as a preparation method and a vaccine thereof, and relates to the technical field of biological medicines. The avian influenza HA-Fd fusion protein sequentially contains an avian influenza HA fragment and a bacteriophage Fibritin foldon fragment from an N end to a C end, the amino acid sequence of the avian influenza HA fragment is shown as SEQ ID NO. 1, and the amino acid sequence of the bacteriophage Fibritin foldon fragment is shown as SEQ ID NO. 2. The avian influenza HA-Fd fusion protein has a trimer conformation, andthe protein structure is close to that of natural protein.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an avian influenza HA-Fd fusion protein, a preparation method and a vaccine thereof. Background technique [0002] Highly pathogenic avian influenza (HPAI) is a severe zoonotic infectious disease caused by influenza A virus. Poultry, especially chickens, are the main source of infection of this infectious disease. In 1997, Hong Kong, China first reported poultry and human infection with highly pathogenic avian influenza H5N1, and then highly pathogenic avian influenza spread widely in the world, causing more than 500 human infections. The mortality rate of highly pathogenic avian influenza is as high as 60%, which poses a continuous threat to human public health. [0003] Influenza A virus belongs to the Orthomyxoviridae family and is an enveloped single-negative-strand RNA virus. Its genome contains 8 independent gene segments with a total length of about 13 kb, which can e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/145A61P31/16
CPCA61K39/12A61K2039/552A61P31/16C07K14/005C07K2319/00C12N15/85C12N2760/16122C12N2760/16134C12N2795/10122
Inventor 贺笋张国庆于新茂聂祥祥孔楚心张莹周冰心李健友李延涛潘毅平
Owner 天康制药股份有限公司
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