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 expressio

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

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

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

[0048] 1, COVID-19 S-RBD fusion protein sequences of the synthetic gene, construction of expression plasmid

[0049] Synthetic S-RBD fusion protein gene sequence (including the front and rear respectively endonuclease Nde I and EcoR I restriction site), which DNA sequence, corresponding to the amino acid sequence shown as SEQ ID No.1 by SEQ ID No.2 Show. The S-RBD was cloned into pET-28a vector, protein C terminal strip 6 histidines to facilitate purification late; monoclonal screened positive for the correct expression in E. coli expression system; wherein, size of the protein S-RBD is 30kDa.

[0050] 2, recovery puncture bacteria

[0051] Take a clean pipette tip, growth lines along the piercing tube is inserted into E. coli as a pale yellow, the operation is repeated several times back and forth, each time a slight change in direction of the pipette tip, the pipette tip to increase the conta...

Example Embodiment

[0083] Preparation COVID-19 S-RBD protein colloidal gold card detection reagents: Example 2

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

[0085] (1) Coating: 1ml of colloidal gold, was added K 2 C0 3 , Was added S-RBD protein, rotary mixing device mixing 15min; 1ml Another colloidal gold, was added K 2 C0 3 , Then add chicken IgY antibody, rotary mixing device mixing 15min;

[0086] (2) Blocking: BSA were added for blocking the rotary mixing device mixing 15min; 6000rpm 10min centrifugation supernatant, centrifugation was repeated 10min, the supernatant, were added to 100μl of pure water to give a working solution of colloidal gold and colloidal gold fluid 2.

[0087]2. Preparation of the plasma gold pad: mix the colloidal gold working fluid 1 and the colloidal working fluid 2, dilute the dilution (PBS + 2% trehalose), and the volume of the colloidal gold working fluid 1 is added. Finally, 1 / 8 of the golden dilution volume, the volume of the colloidal gold working fluid 2...

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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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