CN111647053A

CN111647053A - Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening

Info

Publication number: CN111647053A
Application number: CN202010300151.4A
Authority: CN
Inventors: 于晓波; 张晓梅; 王聃; 王红叶; 梁特; 代佳宇
Original assignee: Institute Of Life Sciences Academy Of Military Medicine Academy Of Military Sciences
Current assignee: Institute Of Life Sciences Academy Of Military Medicine Academy Of Military Sciences
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2020-09-11

Abstract

The invention provides a polypeptide for 2019 novel coronavirus (SARS-CoV-2) detection, antibody or vaccine drug screening, wherein the polypeptide comprises at least 15 continuous amino acid sequences in S protein, N protein or orf1ab protein sequences of SARS-CoV-2 virus proteins, and the polypeptide comprises any one amino acid sequence shown in SEQ ID No. 1-25.

Description

Polypeptide and application thereof in novel coronavirus detection and antibody or vaccine screening

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a polypeptide and application thereof in 2019 novel coronavirus (SARS-CoV-2) detection and antibody or vaccine drug screening.

Background

Since 12 months in 2019, many viral pneumonia cases are found in various parts of the world, and the diagnosis results are all viral pneumonia. The world health organization formally named the novel coronavirus 2019 (SARS-CoV-2) at 11/2/2020, and the similarity with the SARS virus reaches 82%. However, the novel virus still has no effective treatment method, has strong infectivity, wide dissemination range and long hiding time, has few virus infected patients without disease symptoms or with light symptoms, and can induce secondary or even tertiary transmission if people who are infected and contacted with the virus are not controlled in time. In addition, without the development of an effective vaccine, new pneumovirus may be present in humans for a long time.

The SARS-CoV-2 virus proteome is composed of a plurality of proteins, Open Reading Frame (ORF) ORF1ab protein, four structural proteins E protein (envelope), M protein (membrane), N protein (nucleocapsid), S protein (Spike), five auxiliary proteins ORF3a, ORF6, ORF7a, ORF8, ORF 10. Orf1ab is involved in viral RNA replication and transcription. The E and M proteins are important for viral assembly of coronaviruses, and the N protein is an essential protein for the synthesis of viral RNA.

The patent CN202010215184.9 (application date: 2020-03-24) that the inventor of the present invention has applied for describes that all the coding protein sequences of SARS-CoV-2 are extracted from NCBI data, and SARS-CoV-2 virus proteome polypeptide chips are designed and prepared, so as to implement panoramic scan of all SARS-CoV-2 virus antibodies in the blood of the patient infected with new type pneumonia virus, and the whole content of the invention can be used as the content of the present invention.

On the basis of the above patents, the inventor continues to design corresponding polypeptides according to the S protein, N protein and orf1ab protein of 2019 novel coronavirus SARS-CoV-2, and finally determines the epitope by detecting the serum of SARS-CoV-2 virus patient, which can be used for screening and developing SARS-CoV-2 virus antibody or vaccine.

Disclosure of Invention

In a first aspect, the present invention provides a polypeptide comprising at least 15 contiguous amino acid sequences of the protein S, N or orf1ab of the SARS-CoV-2 virus, and any one of the amino acid sequences shown in SEQ ID Nos. 1 to 25 listed in Table 1.

TABLE 1

Preferably, the protein N sequence of the SARS-CoV-2 virus protein is shown as SEQ ID No.34, the protein sequence of orf1ab is shown as SEQ ID No.26, and the protein sequence of S is shown as SEQ ID No. 27.

Preferably, the polypeptide comprises at least 15-355 consecutive amino acids at position 751-1105 of the S protein sequence of SARS-CoV-2 virus protein, for example, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 355 consecutive amino acids. In the embodiment of the present invention, the polypeptide is 15-355 consecutive amino acids at the 751-1105 th position of the S protein sequence of SARS-CoV-2 virus protein.

Preferably, the polypeptide comprises at least 15-25 consecutive amino acids from 81-105 of the N protein sequence of SARS-CoV-2 virus protein, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 consecutive amino acid sequences. In the embodiment of the invention, the polypeptide is 15-25 continuous amino acids at positions 81-105 of an N protein sequence of SARS-CoV-2 virus protein.

Preferably, the polypeptide comprises 15 continuous amino acid sequences at 3361-3375 of the protein sequence orf1ab of SARS-CoV-2 virus protein.

In a specific embodiment of the invention, the amino acid sequence of the polypeptide is shown in SEQ ID Nos. 1-25.

Preferably, the polypeptide is obtained by chemical synthesis or prokaryotic expression.

The second aspect of the invention provides the application of the polypeptide in detecting 2019 novel coronavirus SARS-CoV-2, which comprises at least 15 continuous amino acid sequences in SARS-CoV-2 virus protein S protein, N protein or orf1ab protein sequence, and the polypeptide comprises any one of the amino acid sequences shown in SEQ ID No. 1-25.

Preferably, the detection may be an in vivo viral detection in humans or animals, or alternatively, the detection may be a viral detection in an environment (e.g., water, soil, etc.).

Preferably, the detection refers to quantitative or typing detection of the virus.

The third aspect of the invention provides the application of the polypeptide in preparing a product for detecting 2019 novel coronavirus SARS-CoV-2.

Preferably, the product is a biochip, an ELISA kit, a magnetic bead or a cellulose membrane, and the biochip is preferably a polypeptide chip.

The fourth aspect of the invention provides an application of the polypeptide in screening, treating and/or preventing COVID-2019 antibodies or vaccines.

Preferably, the antibody is a neutralizing antibody.

Preferably, the vaccine is a viral vector vaccine, a DNA vaccine, a subunit vaccine, a whole virus inactivated vaccine or an attenuated vaccine.

In a fifth aspect, the invention provides an antibody that binds to a binding epitope of at least one of the S, N or orf1ab protein sequences of the SARS-CoV-2 viral protein.

Preferably, the antibody is an IgM antibody or an IgG antibody.

Preferably, the binding epitope of the S protein of the SARS-CoV-2 virus protein is selected from amino acids 751-1105 of the S protein, and preferably, the binding epitope is selected from at least 15-355 consecutive amino acids 751-1105 of the S protein, for example, including 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 355 consecutive amino acids. In the embodiment of the invention, the binding epitope is selected from amino acids at positions 751-. Preferably, when the antibody is the above-described binding epitope, the antibody is an IgG antibody.

Preferably, the binding epitope of the S protein of the SARS-CoV-2 viral protein is selected from amino acids 811-825 or 981-995 of the S protein. Preferably, when the antibody is the above-mentioned binding epitope, the antibody is an IgM antibody.

Preferably, the binding epitope of the N protein of the SARS-CoV-2 virus protein is selected from amino acids 81 to 105 of the N protein sequence, and preferably, the binding epitope is selected from amino acids 81 to 105 of the N protein and at least 15 to 25 consecutive amino acids, for example, including 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 consecutive amino acid sequences. In an embodiment of the invention, the binding epitope is selected from amino acids 81-95, 91-105 of the N protein. Preferably, when the antibody is the above-mentioned binding epitope, the antibody is an IgM antibody.

Preferably, the binding epitope of orf1ab protein of SARS-CoV-2 virus protein is selected from amino acids 3361-3375 of the orf1ab protein sequence. Preferably, when the antibody is the above-mentioned binding epitope, the antibody is an IgM antibody.

According to a sixth aspect of the present invention there is provided a vaccine comprising a polypeptide or an RNA or DNA sequence encoding said polypeptide, said polypeptide comprising at least 15 contiguous amino acid sequences of the S, N or orf1ab protein sequence of the SARS-CoV-2 viral protein, and said polypeptide comprising any one of the amino acid sequences set forth in SEQ ID Nos. 1 to 25 set forth in Table 1.

Preferably, the amino acid sequence of the polypeptide is shown in SEQ ID No. 1-25.

The seventh aspect of the invention provides a product for detecting SARS-CoV-2 virus, comprising a carrier and a polypeptide distributed on the carrier, wherein the polypeptide sequence at least comprises at least 15 continuous amino acid sequences in S protein, N protein or orf1ab protein sequence of SARS-CoV-2 virus protein, and the polypeptide comprises any one of the amino acid sequences shown in SEQ ID No.1-25 listed in Table 1, and the carrier is a chip, an ELISA plate, a magnetic bead or a cellulose membrane.

Preferably, the product for detecting SARS-CoV-2 virus is a polypeptide chip, which comprises a substrate and a polypeptide distributed on the substrate, at least comprising at least 15 consecutive amino acid sequences in S protein, N protein or orf1ab protein sequence of SARS-CoV-2 virus protein, and the polypeptide comprises any one of the amino acid sequences shown in SEQ ID No.1-25 listed in Table 1.

Preferably, the substrate includes, but is not limited to, glass, silicon wafer, ceramic, mica, metal, plastic, polymer film, etc., preferably glass, silicon wafer and ceramic.

Preferably, the substrate of the present invention is a three-dimensional D-modified glass slide.

Preferably, the substrate of the present invention further comprises a positive control and/or a negative control.

As will be appreciated by those skilled in the art, the polypeptide chip of the present invention may be prepared by conventional methods. In one embodiment of the present invention, the method for preparing the polypeptide chip of the present invention comprises: synthesizing polypeptide, taking the three-dimensional D-modified glass slide as a substrate, and preparing the synthesized polypeptide and/or a positive control spot on the surface of the glass slide by a spot printer.

The eighth aspect of the invention also provides a 2019 novel coronavirus SARS-CoV-2 detection method, which comprises the following steps:

the sample to be detected is contacted with the product for SARS-CoV-2 virus detection, and the fluorescence, chemiluminescence and surface plasma resonance method are adopted for detection.

Preferably, the method for detecting 2019 novel coronavirus SARS-CoV-2 comprises

Sealing the surface of the polypeptide chip; adding a sample to be tested into the polypeptide chip of the invention, and incubating; washing; adding a secondary antibody-fluorescent dye; washing again; scanning was performed with a chip scanner.

Preferably, the chip surface sealing step includes: add 400. mu.l/well 5% milk to the chip and block at room temperature for 0.5-2 h. More preferably, the milk is 0.5g skimmed milk powder added to 10ml PBST (phosphate Tween buffer). More preferably, the blocking time is 1 h.

Preferably, the sample to be detected is serum, and the sample to be detected is 4 mul of serum to be detected, added into 400 mul of 5% milk and mixed evenly. More preferably, the milk is 0.5g skimmed milk powder added to 10ml PBST (phosphate Tween buffer).

Preferably, the sample to be tested is added to the chip and then incubated for 2h at room temperature.

Preferably, the washing is 0.05% PBST washing 1-4 times, 5-15min each time; then ddH2O was washed 1-4 times for 1-5min each. More preferably, the wash is 0.05% PBST wash 3 times, each time for 10min, then ddH2O wash 3 times, each time for 2 min.

Preferably, the secondary antibody in the secondary antibody-fluorescent dye is IgM, IgG or IgA antibody, and more preferably, the secondary antibody is coat-anti-hIgG, coat-anti-hIgA or donkey-anti-hIgG.

Preferably, the fluorescent dye in the secondary antibody-fluorescent dye is Cy3, dye 532, dye 635 or dye 647.

Preferably, the second washing comprises 0.05% PBST washing 1-4 times, each time for 1-10 min; more preferably, the second washing includes 0.05% PBST washing 3 times, each time for 5 min.

The SARS-CoV-2 coronavirus protein described in the invention comprises: rf1ab polyprotein, Sglycoprotein, ORF3a protein, E envelope protein, M structural protein, ORF6protein, ORF7a protein, ORF8protein, N nucleomapped phosphoprotein and ORF10protein, the protein sequence is shown in Table 2 below.

TABLE 2SARS-CoV-2 coronavirus encoded proteins

SEQ ID No.	Name of protein	Abbreviation of protein	Genbank number
				26	orf1ab polyprotein		QHD43415.1
27	surface glycoprotein	S	QHD43416.1
				28	ORF3a protein		QHD43417.1
29	envelope protein	E	QHD43418.1
				30	membrane glycoprotein	M	QHD43419.1
31	ORF6 protein		QHD43420.1
				32	ORF7a protein		QHD43421.1
33	ORF8 protein		QHD43422.1
				34	nucleocapsid phosphoprotein	N	QHD43423.2
35	ORF10 protein		QHI42199.1

Drawings

FIG. 1: box plot analysis of IgG antibody responses of patients with COVID-19 and influenza and non-influenza to different epitopes of the SARS-CoV-2 viral protein.

FIGS. 2 to 23: boxed graph analysis of IgM antibody responses of influenza-CoV-2 patients and influenza and non-influenza patients to different epitopes of SARS-CoV-2 viral protein, in which the polypeptide sequences in FIGS. 2-23 are aa751-765 (FIG. 2), aa761-775 (FIG. 3), aa771-785 (FIG. 4), aa781-795 (FIG. 5), aa791-805 (FIG. 6), aa801-815 (FIG. 7), aa811-825 (FIG. 8), aa821-835 (FIG. 9), aa831-845 (FIG. 10), aa-841 (FIG. 11), aa-875 (FIG. 12), aa871-885 (FIG. 13), aa881-895 (FIG. 14), aa891-905 (FIG. 15), aa901-915 (FIG. 16), aa 911-861 (FIG. 17), aa921-935 (FIG. 18), aa-965 (FIG. 19), aa961-975 (FIG. 20), aa981-995 (FIG. 21), aa991-1005 (FIG. 22), aa1091-1105 (FIG. 23).

Detailed Description

EXAMPLE 1 Synthesis of the polypeptide

Polypeptide sequences were designed according to table 1, and the synthesis steps were as follows from sequence C-terminus to N-terminus:

1) weighing 0.3g of resin, putting the resin into a glass reactor, adding DCM (dichloromethane) and swelling for 30 minutes;

2) removing DCM, adding 0.6g of the first amino acid in the amino acid sequence, adding 0.6g of DIEA (diisopropylethylamine), DMF (dimethylformamide) and DCM, carrying out nitrogen bubbling reaction for 60 minutes, removing the reaction solution, adding DMF and MEOH, and washing for three times;

3) the second amino acid 0.6g in the amino acid sequence was added to the reactor, HBTH (1-hydroxy-benzotrichloride tetramethylhexafluorophosphate) and DIEA were added, the reaction was bubbled with nitrogen for 30 minutes, the liquid was washed off, ninhydrin detection was performed, and then end capping with pyridine and acetic anhydride was performed. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin;

4) the amino acids in table 1 were added in sequence according to step 3);

5) and taking down the reaction column after the resin is dried by nitrogen. Transferring to a10 mL centrifuge tube, adding 6mL cutting fluid (95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water), shaking and filtering off the resin;

6) adding a large amount of diethyl ether into the filtrate to separate out a crude product, centrifuging, and cleaning to obtain the crude product;

7) purifying the polypeptide, namely purifying the crude product to 90% by high performance liquid chromatography;

8) freeze-drying the polypeptide, concentrating the purified liquid in a freeze dryer, and freeze-drying to obtain white powder.

EXAMPLE 2 preparation of polypeptide chips

Selecting a three-dimensional D modified glass slide as a substrate of the polypeptide chip;

all the polypeptide fragments synthesized in example 1 and positive control spots were prepared on the surface of a slide by a microarray spotter.

EXAMPLE 3 polypeptide chip test of the effectiveness of detecting SARS-CoV-2 protein antibodies

Collecting samples:

samples were obtained from Beijing Hospital and were diagnosed in patients with mild symptoms of COVID-19 according to the "diagnosis and treatment protocol for pneumonia with novel coronavirus infection" (test version 7). 15 patients with early stage COVID-19, 13 patients with influenza with similar symptoms and 12 patients with non-influenza.

Screening of serum samples:

1. and (3) sealing: the chip is added with a fence, and 400ul/well 5% milk (0.5g milk powder +10ml PBST) is added to be sealed for 0.5h at room temperature;

2. sample preparation: mixing 4ul serum and 400ul 5% milk;

3. sample adding: adding the diluted serum sample into the polypeptide chip prepared in the implementation 2, incubating for 1h at room temperature in a manner of 400 ul/well;

4. washing: washing with 0.05% PBST for 3 times, each for 10 min;

5. adding a fluorescent dye: adding Donkey-anti-hIgG-532 and coat-anti-hIgM 635 (original concentration is 1mg/ml, 4.8ul +1200ul mil) into the chip array, and incubating for 0.5h at room temperature in a dark place;

6. washing in dark: washing with 0.05% PBST for 5min 3 times; then ddH2O was washed 2 times for 2min each;

7. chip detection, drying the chip and using

4300A chip scanner 532nm, 635nm channel scan chip, use software GenePixpro 6.0(Molecular Devices, CA, USA) to extract chip fluorescence signal value.

Statistical analysis:

all microarray signals were normalized with z-score before statistical analysis. Differentially expressed SARS-CoV-2 antibodies were identified using the MannWhitney u test with a p-value of 0.05.

The experimental results are as follows:

the SARS-CoV-2 virus polypeptide chip is used to analyze the level of antibodies binding to epitopes of viral proteins in serum samples from neocoronary patients and influenza and non-influenza suspected patients. As shown in fig. 1-23, antibody levels were significantly elevated for 22 IgM antigens and 5 IgG antigens in COVID-19 compared to the influenza and non-influenza groups (P < 0.05).

While the invention has been described in conjunction with specific embodiments thereof, it is to be understood that both the foregoing description and the following description are intended to provide a better understanding of the invention, and are not intended to limit the invention in any way. Those skilled in the art, having read the present specification, may make necessary alterations to the particular embodiments of the invention without departing from the spirit and scope of the invention. The scope of the invention is defined by the appended claims, and all equivalents to the claims are intended to be embraced therein.

Claims

1. A polypeptide comprising at least 15 contiguous amino acid sequences of the S, N or orf1ab protein sequence of a SARS-CoV-2 viral protein, and comprising any one of the amino acid sequences set forth in SEQ ID nos. 1-25.

2. The polypeptide of claim 1, wherein the polypeptide comprises at least 15-355 consecutive amino acids at positions 751-1105 of the S protein sequence of SARS-CoV-2 virus protein, or the polypeptide comprises at least 15-25 consecutive amino acids at positions 81-105 of the N protein sequence of SARS-CoV-2 virus protein, or the polypeptide comprises 15 consecutive amino acids at positions 3361-3375 of the orf1ab protein sequence of SARS-CoV-2 virus protein.

3. A polypeptide according to claim 1, having an amino acid sequence as shown in any one of SEQ ID Nos. 1 to 25.

4. The application of the polypeptide in preparing a product for detecting 2019 novel coronavirus SARS-CoV-2, wherein the polypeptide comprises at least 15 continuous amino acid sequences in SARS-CoV-2 virus protein S protein, N protein or orf1ab protein sequences, and the polypeptide comprises any one of the amino acid sequences shown in SEQ ID No. 1-25.

5. The use according to claim 4, wherein the product is a biochip, an ELISA kit, a magnetic bead or a cellulose membrane.

6. Use of a polypeptide comprising at least 15 contiguous amino acid sequences of the protein S, N or orf1ab of the SARS-CoV-2 virus and comprising any one of the amino acid sequences shown in SEQ ID nos. 1-25 in the screening of antibodies or vaccines for the treatment and/or prevention of covi-2019.

7. An antibody, wherein the binding epitope of the antibody and the S protein of the SARS-CoV-2 virus protein is selected from the amino acids at the 751-1105 th site of the S protein, preferably the binding epitope is selected from the amino acids at the 751-1105 th site, the 761-775, the 771-785, the 781-795, the 791-805, the 801-815, 811-825, 821-835, 831-845, 841-855, 861-875, 871-885, 881-895, 891-905, 901-915, 911-925, 921-935, 951-965, 961-975, 981-995, 991-1105 1005 or 1091-cystine or,

the binding epitope of the antibody and the N protein of SARS-CoV-2 virus protein is selected from amino acids 81-105 of the N protein sequence, preferably, the binding epitope is selected from amino acids 81-95, 91-105 of the N protein, or,

the binding epitope of the antibody and the orf1ab protein of the SARS-CoV-2 virus protein is selected from amino acids 3361-3375 of the orf1ab protein sequence.

8. A vaccine comprising a polypeptide comprising at least 15 contiguous amino acid sequences of the S, N or orf1ab protein sequences of the SARS-CoV-2 viral protein, and any one of the amino acid sequences set forth in SEQ ID nos. 1-25, or RNA or DNA encoding said polypeptide.

9. A product for detecting SARS-CoV-2 virus comprises a carrier and polypeptide distributed on the carrier, the polypeptide comprises at least 15 continuous amino acid sequences in S protein, N protein or orf1ab protein sequence of SARS-CoV-2 virus protein, and the polypeptide comprises any one amino acid sequence shown in SEQ ID No.1-25, the carrier is chip, ELISA plate, magnetic bead or cellulose membrane.

10. A2019 method for detecting novel coronavirus SARS-CoV-2, the method comprises:

contacting a sample to be tested with the product of claim 9, and detecting by fluorescence, chemiluminescence, surface plasmon resonance.