Recombinant novel coronavirus COVID-19S-RBD protein and preparation method and application thereof

A technology for COVID-19S-RBD and coronavirus, which is applied in the field of recombinant novel coronavirus COVID-19S-RBD protein and its preparation, can solve the problems affecting the antigenicity of recombinant protein, complex production process, and reduced detection performance, so as to achieve convenient Effective control of the epidemic, high protein expression, and easy operation

Active Publication Date: 2020-12-15
SHANGHAI JIEMEN BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, animal cells such as HEK-293 and CHO cells are usually chosen to express recombinant proteins, but the protein expression of these cells is low, and the production process is complicated and the cost is high
Although the recombinant protein expressed in the E. coli expression system has a high expression level and is easy to operate, there may be large differences between the protein folding and modification and the natural protein after translation, and this difference will affect the antigenicity of the recombinant protein. , reducing its detection performance

Method used

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  • Recombinant novel coronavirus COVID-19S-RBD protein and preparation method and application thereof
  • Recombinant novel coronavirus COVID-19S-RBD protein and preparation method and application thereof
  • Recombinant novel coronavirus COVID-19S-RBD protein and preparation method and application thereof

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Embodiment 1

[0047] Embodiment 1: Preparation of recombinant novel coronavirus COVID-19 S-RBD protein

[0048] 1. Synthesis of COVID-19 S-RBD fusion protein gene sequence and construction of expression plasmid

[0049] Artificially synthesized S-RBD fusion protein gene sequence (respectively containing endonuclease Nde I and EcoR I restriction sites before and after), its DNA sequence is shown in SEQ ID No.1, and the corresponding amino acid sequence is shown in SEQ ID No.2 Show. The S-RBD gene was cloned into the pET-28a vector, so that 6 histidines were attached to the C-terminus of the protein to facilitate later purification; the correct positive single clone was screened for expression in the E. coli expression system; among them, the size of the S-RBD protein was is 30kDa.

[0050] 2. Recovery of puncture bacteria

[0051] Take a clean pipette tip, insert it along the pale yellow E. coli growth line in the puncture tube, and repeat the operation several times, changing the directi...

Embodiment 2

[0083] Embodiment 2: Preparation of COVID-19 S-RBD protein colloidal gold detection reagent card

[0084] 1. S-RBD protein coated nano colloidal gold

[0085] (1) Coating: take 1ml colloidal gold, add K 2 C0 3 , then add S-RBD protein, and mix with a rotary mixer for 15 minutes; take another 1ml colloidal gold, add K 2 C0 3 , then add the chicken IgY antibody, and mix with a rotary mixer for 15 minutes;

[0086] (2) Sealing: add BSA to seal, and mix with a rotary mixer for 15 minutes; centrifuge at 6000rpm for 10 minutes to take the supernatant, repeat centrifugation for 10 minutes, take the supernatant, add 100 μl pure water to obtain colloidal gold working solution 1 and colloidal gold working solution 2.

[0087]2. Preparation of colloidal gold pad: mix colloidal gold working solution 1 and colloidal gold working solution 2, dilute with diluent (PBS+2% trehalose) to make gold paving dilution, add colloidal gold working solution 1 to a volume of 1 / 8 of the volume of th...

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Abstract

The invention discloses a recombinant novel coronavirus COVID-19S-RBD protein and a preparation method and application thereof. The preparation method comprises the steps that an artificially synthesized S-RBD fusion protein gene is constructed into an escherichia coli expression system, and then the soluble purified recombinant COVID-19S-RBD protein with high expression quantity and antigenicityis obtained through a special inclusion body renaturation technology. The preparation method of the recombinant protein is easy and convenient to operate, low in production cost and high in efficiency, and the prepared recombinant protein can be used for preparing a reagent strip for detecting new coronavirus; the using effect of the reagent strip is verified by the preparation method, the detection result is accurate, the efficiency is high, the result is popular and easy to understand, and the preparation method is particularly suitable for rapid screening of the new coronavirus of a large number of people.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant novel coronavirus COVID-19 S-RBD protein and its preparation method and application. Background technique [0002] Coronavirus is a large family of viruses that are important pathogens of many domestic animals, pets, including human diseases, and can cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). [0003] Coronaviruses belong to the genus Coronavirus of the family Coronaviridae in the taxonomic category. The novel coronavirus (COVID-19) belongs to the Sarbecovirus subgenus of βcoronavirus, and is a kind of RNA virus with an envelope and a linear single-strand positive strand genome. Its particles are round or oval, and often pleomorphic. The diameter is 60~140nm. The clinical symptoms of the new coronavirus infection are similar to those of SARS virus. In terms of gene seq...

Claims

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Application Information

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IPC IPC(8): C07K14/165C12N15/70C07K1/22C07K1/36C07K1/34G01N33/569G01N33/558G01N33/543G01N33/532
CPCC07K14/005C12N15/70G01N33/56983G01N33/558G01N33/532G01N33/543C12N2770/20022C12N2770/20051G01N2333/165
Inventor 付理文刘艳慧娄珺周伟刘容
Owner SHANGHAI JIEMEN BIO TECH
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