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Recombinant fusion protein containing arctic ground squirrel hepatitis virus core protein, preparation method and applications thereof

A fusion protein and hepatitis virus technology, applied in the field of recombinant fusion protein and its preparation, can solve problems such as hidden safety hazards and weak vaccine immune response, and achieve the effects of easy purification and high protein expression

Active Publication Date: 2017-07-28
NAT VACCINE & SERUM INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a recombinant fusion protein containing the core protein of arctic ground squirrel hepatitis virus and its preparation method and application in view of the weak immune response and potential safety hazards of some vaccines in the prior art

Method used

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  • Recombinant fusion protein containing arctic ground squirrel hepatitis virus core protein, preparation method and applications thereof
  • Recombinant fusion protein containing arctic ground squirrel hepatitis virus core protein, preparation method and applications thereof
  • Recombinant fusion protein containing arctic ground squirrel hepatitis virus core protein, preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Construction and identification of recombinant fusion protein yeast expression plasmid

[0100] 1. Design of recombinant fusion protein

[0101] Taking the amino acid sequence of the Arctic ground squirrel hepatitis virus core protein (UniProtKB / Swiss-Prot:Q64897.1) as the reference sequence, the carrier sequence as shown in SEQ ID NO.1 is formed, at the 78th and The 254-277th amino acid epitope (shown in SEQ ID NO.3) of the respiratory syncytial virus fusion protein (Genebank: ACO83301.1) is inserted between the 79th amino acid, which is the binding position of Palivizumab , formed in series through the "GILE" and "L" amino acid linking arms to form the amino acid sequence shown in SEQ ID NO.2.

[0102] 2. Gene optimization and synthesis

[0103] According to the amino acid sequence of the recombinant fusion protein shown in SEQ ID NO.2, the gene sequence was optimized according to the pros and cons of Hansenula codons and the abundance of tRNA to form the...

Embodiment 2

[0110] Example 2: Screening and identification of highly expressed positive yeast strains

[0111] 1. Conversion

[0112] NVSI-H.P-105 (△URA3△LEU2) Hansenula spp. was cultured in YPD liquid medium. When the cell density (OD600) reached 1.0, the preparation of yeast competence was carried out. The large fragment gene in the final PUC25-AGRU plasmid was transformed into NVSI-H.P-105 yeast by electroporation, and finally the transformed bacteria liquid was spread on the SM-leu solid medium and cultured at 37°C for 3-5 days. Obtain transformed recombinants.

[0113] 2. ELISA screening

[0114] Pick the monoclonal colonies grown on the SM-leu solid medium and place them in 2ml SM-leu liquid medium for cell culture, and culture them at 37°C and 250rpm for 24 hours with shaking. Take 200 μl of bacterial liquid and transfer it to 4ml SM-leu liquid medium to continue culturing. After the bacterial cell density (OD600) reaches above 10, harvest the bacterial cells by centrifugation a...

Embodiment 3

[0124] Example 3: Preparation and identification of cVLP

[0125] 1. Yeast fermentation and cell crushing

[0126] The recombinant fusion protein high-expression yeast strain screened in Example 2 was inoculated in 10ml of MD liquid medium for shaking culture for 24 hours, then transferred to 100ml of MD liquid medium and expanded for 24 hours to prepare fermented seeds, and inoculated in Yeast fermentation is carried out in a 5L fermenter, and methanol is used to induce expression of the target protein. After the fermentation, the cells were washed twice with normal saline, and finally the cells were resuspended in a crushing buffer (20mM PB, 50mM NaCl, pH 7.2) for high-pressure crushing, and the protein supernatant was obtained by centrifugation. The whole bacterial protein before and after induction was subjected to 10% SDS-PAGE electrophoresis analysis, and the results were as follows: Figure 4 As shown in A, the arrow points to the target protein band.

[0127] 2. Tar...

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Abstract

The present invention provides a recombinant fusion protein containing an arctic ground squirrel hepatitis virus core protein, wherein the recombinant fusion protein comprises an arctic ground squirrel hepatitis virus core protein and an exogenous protein fragment inserted between the amino acids of the arctic ground squirrel hepatitis virus core protein. According to the present invention, the arctic ground squirrel hepatitis virus core protein is firstly used as the exogenous epitope presentation vector; and by optimizing the design, the recombinant fusion protein is expressed in Hansenula polymorpha to form the chimeric virus-like particles, the chimeric virus-like particles adopted as the vaccine component can stimulate to produce strong immune response under the premise of the assurance of the safety of the vaccine, and the chimeric virus-like particles can be used for preventing RSV infection, and have important scientific and practical value.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant fusion protein comprising arctic ground squirrel hepatitis virus core protein and its preparation method and application. Background technique [0002] Respiratory syncytial virus (RSV) is the main pathogen causing lower respiratory tract infection in infants and young children worldwide. More than 90% of infants experience at least one RSV infection within two years after birth, and infants under 6 months, the elderly and immunocompromised population are prone to severe infection and death. For diseases caused by RSV infection, the only effective marketed drug is palivizumab (Palivizumab). RSV belongs to the family Paramyxoviridae, the genus Pneumovirus, and is a non-segmented single-stranded negative-sense RNA enveloped virus. The virus genome is 15.2kb in full length and contains 10 coding genes (NS1, NS2, N, P, M, SH, G, F, M2, L in sequence), encoding 11 proteins, ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/81C12N7/04C07K16/10C12N1/19A61K39/155A61P31/14C12R1/78C12R1/93
CPCA61K39/12A61K2039/5258A61K2039/55505C07K14/005C07K16/1027C07K2319/00C12N7/00C12N15/62C12N15/815C12N2730/10122C12N2730/10123C12N2760/18522C12N2760/18534C12N2800/22
Inventor 李启明梁宇邵帅靳玉琴陈实张靖侯俊伟唐芳张学峰杜丽芳唐玉龙栗子谦王擎擎
Owner NAT VACCINE & SERUM INST
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