CN113666988A - Preparation method and application of N-terminal structural domain of novel coronavirus nucleocapsid protein - Google Patents
Preparation method and application of N-terminal structural domain of novel coronavirus nucleocapsid protein Download PDFInfo
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Abstract
The invention discloses a preparation method of a novel coronavirus nucleocapsid protein N-terminal structural domain, wherein the amino acid sequence of the protein is shown as SEQ ID N0.1, and the preparation method comprises the following steps: s1, predicting: predicting the amino acid sequence of the N-terminal domain of the protein; s2, expression: expressing the protein in a soluble expression mode of escherichia coli; s3, purification: purifying the protein by a nickel affinity column combination, hybrid protein washing and elution method, namely obtaining a target protein crude product; s4, fine purification: and further purifying the target protein by using a molecular sieve, and removing foreign proteins to obtain the high-purity N-terminal domain of the 2019 novel coronavirus nucleocapsid protein. The invention can quickly obtain the required antigen in the face of outburst epidemic situation, and prepare sufficient antigen in a week with low cost for research and development of diagnostic reagents.
Description
Technical Field
The invention relates to the technical field of biotechnology detection, in particular to a preparation method and application of a novel N-terminal domain of a coronavirus nucleocapsid protein.
Background
Pneumonia infected by the novel coronavirus 2019-n-CoV is mainly manifested by fever, hypodynamia, dry cough and the like, and a few patients are accompanied by upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. Severe cases often develop dyspnea after one week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, refractory metabolic acidosis, and hemorrhagic coagulation dysfunction. The COVID-19 caused by the novel coronavirus 2019-n-CoV has no specific medicine so far. Some cases report that the treatment medicine still needs more clinical practice to prove the effect. The research on effective diagnostic reagents, early diagnosis, early isolation and cut-off of propagation paths are helpful for controlling epidemic spread.
The novel coronavirus 2019-n-CoV nucleic acid detection method has high specificity and sensitivity, but the detection method has high technical requirements, false negative easily occurs, samples need special treatment, professional instrument equipment such as a PCR amplification instrument and gel electrophoresis is required, the detection time of the novel coronavirus is long, the detection result needs to be operated and judged by professional technicians, and the method cannot be applied to early primary screening of communities, primary hospitals, airports, customs, even families and other primary bases.
Therefore, a diagnostic reagent for detecting the infection of the novel coronavirus 2019-N-CoV at an earlier stage, more accurately, more quickly and more effectively is urgently needed for carrying out early differential diagnosis, and the antibody rapid diagnostic reagent is in great demand, and the antibody detection needs to prepare an antigen with antigen-antibody interaction.
Disclosure of Invention
The present invention aims to provide a preparation method and application of a novel N-terminal domain of a coronavirus nucleocapsid protein, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing a novel N-terminal structural domain of a coronavirus nucleocapsid protein, wherein the amino acid sequence of the protein is shown as SEQ ID N0.1, comprises the following steps:
s1, predicting: predicting the amino acid sequence of the N-terminal domain of the protein;
s2, expression: expressing the protein in a soluble expression mode of escherichia coli;
s3, purification: purifying the protein by a nickel affinity column combination, hybrid protein washing and elution method, namely obtaining a target protein crude product;
s4, fine purification: further purifying the target protein by a molecular sieve, and removing foreign proteins to obtain a high-purity 2019 novel coronavirus nucleocapsid protein N-terminal structural domain;
in S3, the method for purifying by using a nickel affinity column, the purification of the target protein comprises three steps:
the method comprises the following steps: the disrupted supernatant was applied to a 1 ml nickel column using a solution of 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, pH 7.4;
step two: the removal of the hetero-proteins was washed with a solution of 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, 10 mmol/l imidazole, pH 7.4.
Step three: elution with 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, 250 mmol/l imidazole, pH 7.4 gives crude target protein.
Preferably, the escherichia coli comprises escherichia coli codon optimized gene synthesis, escherichia coli transformation and expression, escherichia coli bacteria breaking and escherichia coli supernatant obtaining.
Preferably, in step S4, the resulting crude product is further purified by molecular sieves.
Preferably, the fraction at the position of the absorption peak is collected using a molecular sieve column Superdex 20016/60 at a flow rate of 1 ml/min, and run gel analysis confirms the target molecule and purity.
Preferably, in step S4, the purity of the target protein is greater than 90%.
An application of a novel N-terminal structural domain protein of a coronavirus nucleocapsid protein in a 2019 novel coronavirus diagnostic reagent.
The core raw material of the antibody detection rapid diagnostic reagent is an antigen, and the N-terminal domain of the 2019 novel coronavirus nucleocapsid protein is a good target molecule.
The amino acid sequence of the 2019 novel coronavirus nucleocapsid protein N-terminal domain provided by the invention is shown as SEQ ID N0.1, and the preparation method is realized by the following technical scheme: cloning 2019 gene sequence of N-terminal structural domain of novel coronavirus nucleocapsid protein; the synthesized gene sequence is inserted into a pET28a expression vector, the sequence is optimized according to the preference of an escherichia coli codon, the expression vector is used for transforming BL21(DE3), BL21(DE3) is an engineered cell, the cell is used for expressing a large amount of target protein in vitro, a monoclonal is selected, the cell is cultured, the target protein is expressed by induction, then bacteria are broken, the supernatant of a bacterial liquid is collected by centrifugation, and the N-end structural domain of the 2019 novel coronavirus nucleocapsid protein is obtained by purifying through a nickel affinity column, a molecular sieve column and the like.
The 2019 novel coronavirus nucleocapsid protein N-terminal domain is used in a reagent for detecting the novel coronavirus antibody by a colloidal gold method, the 2019 novel coronavirus nucleocapsid protein N-terminal domain protein marked by colloidal gold is used on a gold pad of a reagent strip, and an anti-human antibody marking line is used as a T line.
Compared with the prior art, the invention has the beneficial effects that:
the invention can quickly obtain the required antigen in the face of outburst epidemic situation, and prepare sufficient antigen in a week with low cost for research and development of diagnostic reagents.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a flow chart of a method of making an embodiment of the present invention;
FIG. 3 shows SDS-PAGE analysis of 2019 purification of the N-terminal domain protein of the novel coronavirus nucleocapsid protein.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The preparation method of the N-terminal domain of the novel coronavirus nucleocapsid protein of the embodiment has an amino acid sequence shown as SEQ ID N0.1, and comprises the following steps:
s1, predicting: predicting the amino acid sequence of the N-terminal domain of the protein;
s2, expression: expressing the protein in a soluble expression mode of escherichia coli;
s3, purification: purifying the protein by a nickel affinity column combination, hybrid protein washing and elution method, namely obtaining a target protein crude product;
s4, fine purification: further purifying the target protein by a molecular sieve, and removing foreign proteins to obtain a high-purity 2019 novel coronavirus nucleocapsid protein N-terminal structural domain;
in S3, the method for purifying by using a nickel affinity column, the purification of the target protein comprises three steps:
the method comprises the following steps: the disrupted supernatant was applied to a 1 ml nickel column using a solution of 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, pH 7.4;
step two: the removal of the hetero-proteins was washed with a solution of 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, 10 mmol/l imidazole, pH 7.4.
Step three: elution with 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, 250 mmol/l imidazole, pH 7.4 gives crude target protein.
The escherichia coli of the embodiment comprises escherichia coli codon optimized gene synthesis, escherichia coli transformation and expression, escherichia coli bacteria breaking and escherichia coli supernatant obtaining.
In step S4 of this example, the crude product obtained was further purified by molecular sieves.
In this example, a molecular sieve column Superdex 20016/60 was used at a flow rate of 1 ml/min, and fractions at the absorption peak positions were collected and run gel analysis was performed to confirm the target molecule and purity.
In step S4 of this example, the purity of the target protein is greater than 90%.
The application of the N-terminal domain protein of the novel coronavirus nucleocapsid protein of the embodiment in 2019 novel coronavirus diagnostic reagents.
As shown in fig. 1, according to an exemplary embodiment of the present invention, there is provided a method for preparing an N-terminal domain of a 2019 novel coronavirus nucleocapsid protein, the amino acid sequence of the protein is shown as SEQ ID N0.1, the method comprising the steps of:
s1 predicts: predicting the amino acid sequence of the receptor binding domain of said protein;
s2 expresses: expressing the protein in a mode of expressing the inclusion body of the escherichia coli;
s3 purification: purifying the protein by an affinity column and a hybrid protein washing method to obtain a crude target protein product;
s4 fine purification: and further purifying the crude product by a molecular sieve to remove foreign proteins, thus obtaining the highly purified N-terminal domain of the 2019 novel coronavirus nucleocapsid protein.
As shown in fig. 2, according to an exemplary embodiment of the present invention, there is provided a method for preparing an N-terminal domain of a 2019 novel coronavirus nucleocapsid protein, the method comprising the steps of:
firstly, predicting the N-terminal structural domain of the 2019 novel coronavirus nucleocapsid protein, selecting a sequence shown as an amino acid sequence table SEQ ID N0.1, synthesizing a gene sequence optimized by an escherichia coli codon, and constructing an expression vector pET28 a;
transforming escherichia coli BL21(DE3) competent cells by using the expression plasmid, and screening successfully transformed cells by using antibiotics;
selecting a monoclonal, inoculating the monoclonal into a few milliliters of culture medium, growing for 10 hours, and preparing glycerol bacteria to facilitate subsequent culture;
culturing a larger amount of cells until the optical density reaches about 0.75, adding an inducer isopropyl-beta-D-thiogalactose with the final concentration of 1 millimole/liter, inducing the expression of the target protein, and culturing at 37 ℃ for 4 hours.
Collecting cells, crushing and purifying target protein, mainly centrifugally collecting the cells, crushing thalli, and centrifugally collecting supernatant after bacterial crushing.
Preparing a nickel column, washing and equilibrating the column with double distilled water and a buffer solution respectively, centrifuging the supernatant, passing the supernatant through the column at a flow rate of 1-2 ml/min, washing the hybrid protein with a buffer solution containing 20 mmol/l imidazole, and eluting with a buffer solution containing 250 mmol/l imidazole;
further purification of the target protein by the molecular sieve: the eluted solution is further concentrated to about 5 mg/ml, and 1-2 ml of the solution is loaded to Superdex 20016/60 at a flow rate of 1 ml/min, and absorption peaks are collected.
And (3) analyzing the purity and the expression quantity by SDS-PAGE running gel, wherein the purity reaches more than 90 percent, and the expression quantity of one liter can reach hundreds of milligrams. The purification of the N-terminal domain of the novel coronavirus nucleocapsid protein 2019 as shown in FIG. 3 by SDS-PAGE analysis.
Sequence listing SEQ ID N0.1:
SDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALAL。
Claims (6)
1. a preparation method of a novel N-terminal structural domain of a coronavirus nucleocapsid protein is characterized in that the amino acid sequence of the protein is shown as SEQ ID N0.1, and the preparation method comprises the following steps:
s1, predicting: predicting the amino acid sequence of the N-terminal domain of the protein;
s2, expression: expressing the protein in a soluble expression mode of escherichia coli;
s3, purification: purifying the protein by a nickel affinity column combination, hybrid protein washing and elution method, namely obtaining a target protein crude product;
s4, fine purification: further purifying the target protein by a molecular sieve, and removing foreign proteins to obtain a high-purity 2019 novel coronavirus nucleocapsid protein N-terminal structural domain;
in S3, the method for purifying by using a nickel affinity column, the purification of the target protein comprises three steps:
the method comprises the following steps: the disrupted supernatant was applied to a 1 ml nickel column using a solution of 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, pH 7.4;
step two: the removal of the hetero-proteins was washed with a solution of 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, 10 mmol/l imidazole, pH 7.4.
Step three: elution with 10 mmol/l tris (hydroxymethyl) aminomethane hydrochloride, 150 mmol/l sodium chloride, 250 mmol/l imidazole, pH 7.4 gives crude target protein.
2. The method for preparing the N-terminal domain of the nucleocapsid protein of the coronavirus as claimed in claim 1, wherein the Escherichia coli comprises the steps of Escherichia coli codon-optimized gene synthesis, Escherichia coli transformation and expression, Escherichia coli disruption and Escherichia coli supernatant acquisition.
3. The method for preparing the N-terminal domain of the nucleocapsid protein of the coronavirus according to claim 1, wherein the crude product obtained in step S4 is further purified by molecular sieve.
4. The method of claim 3, wherein the molecular sieve column Superdex 20016/60 is used at a flow rate of 1 ml/min, and the fractions at the absorption peak position are collected and run gel analysis is performed to confirm the target molecule and purity.
5. The method according to any one of claims 1 to 4, wherein the purity of the target protein in step S4 is greater than 90%.
6. An application of a novel N-terminal structural domain protein of a coronavirus nucleocapsid protein in a 2019 novel coronavirus diagnostic reagent.
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