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Feline coronavirus S recombinant protein and preparation method thereof

A feline coronavirus and recombinant protein technology, applied in botanical equipment and methods, biochemical equipment and methods, viruses, etc., can solve problems such as high variability of S1 functional domains and unfavorable target protein regions

Active Publication Date: 2021-07-06
LONGYAN UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

Among them, the S1 functional domain contains the receptor binding region RBD and a large number of epitope regions, but the S1 functional domain has high variability, which is not conducive to being used as a target protein region; while the S2 functional domain is a relatively conserved part of the S protein, which mediates virus Fusion with cell membrane, and rich in epitopes

Method used

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  • Feline coronavirus S recombinant protein and preparation method thereof
  • Feline coronavirus S recombinant protein and preparation method thereof
  • Feline coronavirus S recombinant protein and preparation method thereof

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preparation example Construction

[0034] The disclosure provides a method for preparing feline coronavirus S recombinant protein, comprising the following steps:

[0035] (a) carrying out PCR amplification with the feline coronavirus S protein gene as a template, and cloning the S recombinant protein fragment;

[0036] The PCR amplification: the upstream primer sequence is shown in SEQ ID NO: 2; the downstream primer sequence is shown in SEQ ID NO: 3; the PCR amplification reaction system is: the amplification system is 25 μL, wherein 2×TransStart FastPfu PCRSuperMix12. 5 μL, 1 μL each of upstream and downstream primers, 1 μL template, supplemented with sterilized ddH 2 O to a final volume of 25 μL; PCR amplification reaction conditions were: pre-denaturation at 94°C for 2 min, followed by cycling, denaturation at 94°C for 20 s; annealing at 55°C for 20 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 5 min.

[0037] (b) The S recombinant protein fragment is separated by electrophoresis,...

Embodiment

[0046] The gene cloning of embodiment feline coronavirus S recombinant protein and the construction of expression vector

[0047] (1) Primer design and synthesis

[0048] Referring to the FCoV S protein gene sequence published in GenBank, a pair of specific primers were designed and synthesized: the upstream primer sequence is shown in SEQ ID NO:2; the downstream primer sequence is shown in SEQ ID NO:3. The double enzyme cutting sites in the downstream primers were designed as NdeI and XhoI respectively. Primers were synthesized, purified and sequenced by Shanghai Sangon Bioengineering Co., Ltd.

[0049] (2) PCR using the above primers

[0050] Feline coronavirus S protein gene was used as template, the amplification system was 25 μL, including 12.5 μL of 2×TransStart FastPfuPCR SuperMix, 1 μL of upstream and downstream primers, 1 μL of plasmid template, supplemented with sterilized ddH 2 O to a final volume of 25 μL. Reaction conditions: Pre-denaturation at 94°C for 2 min...

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Abstract

The invention provides a feline coronavirus S recombinant protein, the amino acid sequence of which is shown as SEQ ID NO: 1. The invention also provides a preparation method of the feline coronavirus S recombinant protein, which comprises the following steps: (a) carrying out PCR (Polymerase Chain Reaction) amplification by taking the gene of the feline coronavirus S protein as a template, and cloning an S recombinant protein fragment; (b) carrying out electrophoretic separation on the S recombinant protein fragment, recovering and purifying, connecting to a vector, and tranforming to competent cells; (c) ligating the plasmid with the prokaryotic expression vector to obtain the recombinant plasmid. and (d) transforming the recombinant plasmid to BL21 for prokaryotic expression to obtain the feline coronavirus S recombinant protein. The inventor finds that the protein fragment in the FCoV S2 protein has good reactogenicity and immunogenicity for the first time, and can be used as a basis for subsequent research and development of FCoV vaccines and related diagnosis and detection kits.

Description

technical field [0001] The disclosure relates to the technical field of gene recombination, in particular to a feline coronavirus S recombinant protein and a preparation method thereof. Background technique [0002] Feline infectious peritonitis is a chronic fatal disease caused by feline coronavirus (FCoV). It is mainly characterized by peritonitis, a large amount of ascites accumulation or granulomatous lesions in various organs. Cats of different ages can be infected, but the elderly The incidence rate of cats and cats under 2 years old is relatively high, and the incidence rate of purebred cats is higher than that of domestic cats of ordinary breeds, and the infection rate is very high. FCoV is a non-segmented, single-stranded positive-sense RNA virus belonging to the order Nidovi-rale, Coronaviridae, and Alphacoronavirus. FCoV has 11 open reading frames (ORFs), encoding 4 structural proteins: spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/50C12N15/70A61K39/215A61P31/14
CPCC07K14/005C12N15/70A61K39/12A61P31/14C12N2770/20022C12N2770/20051C12N2770/20034Y02A50/30
Inventor 林炜明董波吴思研章高强李成钰魏兰张晓东
Owner LONGYAN UNIV
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