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Recombinant plasmid and recombinant bacterium for expressing SARS-CoV-2 N protein, construction method and application

A recombinant plasmid and construction method technology, applied in the field of vaccines, can solve the problems of insufficient activity, loss, difficulty in expression, etc., and achieve the effects of good protein activity, high expression amount, and auxiliary monitoring of infection and outbreak.

Pending Publication Date: 2020-10-16
梁建国
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Problems solved by technology

[0004] At present, it is difficult to express the SARS-CoV-2 N protein in Escherichia coli, because after cloning the coding sequence of the N protein into some expression vectors, it is often found that the expression level meets the requirements, but the activity is not enough or even lost

Method used

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  • Recombinant plasmid and recombinant bacterium for expressing SARS-CoV-2 N protein, construction method and application
  • Recombinant plasmid and recombinant bacterium for expressing SARS-CoV-2 N protein, construction method and application
  • Recombinant plasmid and recombinant bacterium for expressing SARS-CoV-2 N protein, construction method and application

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preparation example Construction

[0031] The present invention has no special restrictions on the source of the full-length sequence of the N protein. During the specific implementation of the present invention, the full-length sequence of the N protein is preferably prepared by the following method: reverse the RNA genome of the SARS-CoV-2 virus Transcribing to obtain cDNA; using the cDNA as a template, using the third primer and the fourth primer to perform PCR amplification to obtain the full-length sequence of the N protein; the present invention has no special method for preparing the RNA genome of the SARS-CoV-2 virus Restrictions are obtained by conventional methods in the art or conventional virus RNA genome kit extracts; the reverse transcription system is calculated in 20 μl and includes the following components: 5×PrimeScript IV cDNA Synthesis Mix 4 μl, Random 6mers (50 μM) 0~ 2μl, RNA genome of SARS-CoV-2 virus: total RNA≤5μg, poly(A)+RNA≤1μg and the rest of RNase Free dH 2 O; the reverse transcrip...

Embodiment 1

[0067] 1. SARS-Cov-2 total cDNA synthesis reaction

[0068] 1) Refer to the reverse transcription system in Table 1 to prepare the following reaction solution on ice:

[0069] Table 1 Reverse transcription system

[0070]

[0071]

[0072] 2) After mixing gently, react according to the following conditions: 30°C for 10 minutes, 95°C for 5 minutes or inactivate the enzyme, then cool on ice.

[0073] 2. N protein gene amplification

[0074] According to the relevant sequence information published in the GenBank database (genome sequence number is MN908947.3), design viral recombinant nucleoprotein (N protein) gene-specific primers (NP forward primer (third primer) is 5'-ATGTCTGATAATGGACCCCAAAATCAG-3', As shown in SEQ ID NO.3; NP reverse primer (the fourth primer) is 5'-TTAGGCCTGAGTTGAGTCAGCAC-3', as shown in SEQ ID NO.4), and carry out PCR amplification to obtain the full-length sequence of N protein, using The PCR product of the full-length sequence of N protein amplif...

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Abstract

The invention provides a recombinant plasmid and recombinant bacterium for expressing SARS-CoV-2 N protein, a construction method and application, and belongs to the technical field of vaccines. The recombinant plasmid takes pET-N-GST-Thrombin-C-His as a skeleton plasmid, and a full-length sequence of a gene for encoding the SARS-CoV-2 N protein is inserted. According to the recombinant plasmid and recombinant bacterium, the SARS-CoV-2 N protein (novel coronavirus nucleoprotein) is expressed by adopting a pET-N-GST-Thrombin-C-His expression vector, the expression quantity of the SARS-CoV-2 N protein is high, and the SARS-CoV-2 N protein obtained by expression can keep good protein activity and can be further applied to a plurality of immunological detection platforms to assist in monitoring infection and outbreak of novel coronavirus.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to a recombinant plasmid expressing SARS-CoV-2 N protein, a recombinant bacterium, a construction method and an application. Background technique [0002] 2019 novel coronavirus (abbreviated as new coronavirus, English name SARS-CoV-2), also known as severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), is a member of the coronavirus family B Type-CoV, a type of severe acute respiratory syndrome-associated coronavirus species, is a positive-sense single-stranded RNA with an envelope, which caused the outbreak of the 2019 novel coronavirus-infected pneumonia (COVID-19) at the end of 2019 . [0003] One of the most important proteins in the nucleocapsid of the new coronavirus is the nucleocapsid protein (N protein). N protein is often used as a diagnostic and detection tool for coronaviruses, and is the core raw m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/50C12N1/21C07K14/165C07K1/22C12R1/19
CPCC12N15/70C07K14/005C12N2770/20022
Inventor 梁建国
Owner 梁建国
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