CN111269313A

CN111269313A - Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit

Info

Publication number: CN111269313A
Application number: CN202010154392.2A
Authority: CN
Inventors: 彭菲; 陈印平
Original assignee: Beijing Yuehao Technology Development Co ltd
Current assignee: Beijing Shunjing Biomedical Technology Co., Ltd
Priority date: 2020-03-07
Filing date: 2020-03-07
Publication date: 2020-06-12
Anticipated expiration: 2040-03-07
Also published as: CN111269313B

Abstract

The invention relates to a monoclonal antibody for detecting novel coronavirus and application of a preparation kit thereof. The N protein is an important marker protein of the coronavirus, and the detection of the N protein can realize the detection of the coronavirus. The anti-N protein monoclonal antibody obtained by the invention has the characteristics of high specificity and mass production, and the titer of the obtained antibody is high. The anti-N protein monoclonal antibody obtained by the invention can be specifically combined with N protein, can be used for immunological detection of cells, has wide market prospect and has important clinical significance.

Description

Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a monoclonal antibody for detecting a novel coronavirus and application of a preparation kit of the monoclonal antibody.

Background

After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. Although no specific treatment method exists for diseases caused by the novel coronavirus, the cure rate of patients with mild diseases or asymptomatic diseases can be greatly improved and the treatment time can be shortened by treating the patients with mild diseases or asymptomatic diseases. Therefore, the detection or identification of the patient becomes particularly important.

Rapid and accurate diagnosis of infectious diseases is a key factor in controlling outbreaks of diseases. With the increasing number of people infected with the novel coronavirus (2019-nCoV), many in vitro diagnostic companies have introduced novel coronavirus detection kits, and many biological companies have competed by providing detection reagents.

There is room for improvement in accuracy and sensitivity of nucleic acid detection. Therefore, antibody detection kits are becoming more important. The novel coronavirus gene encodes a plurality of structural proteins, such as N protein, E protein, S protein and the like, the proteins comprise a plurality of antigen epitopes, and the existence of the antigen can be detected through the antibody by utilizing the principle that the antigen is specifically combined with the antibody, so that the fact that the sample contains the novel coronavirus is directly proved. The antigen of the novel coronavirus can be used as immunogen, plasma cells are stimulated to generate specific antibody after the virus infects human body, and the existence of the antibody can be detected through the antigen by utilizing the principle of the specific combination of the antigen and the antibody, thereby indirectly proving that the human body is infected with the novel coronavirus. Suitable sample types for antibody detection reagents are typically blood, including serum, plasma, and whole blood.

The detected antibodies are mainly divided into IgM and IgG. There is currently no systematic study of the production and duration of these two classes of antibodies to the novel coronaviruses. Usually, IgM antibodies are produced early, rapidly produced upon infection, maintained for a short time, and disappeared rapidly, and detection of positivity in blood can be used as an indicator of early infection. The IgG antibody is produced late, maintained for a long time, disappeared slowly, and the positive detection in blood can be used as the index of infection and past infection.

Therefore, the development of antibody detection kits becomes particularly important.

Disclosure of Invention

The invention provides a coronavirus N protein with better immunogenicity, wherein the nucleotide sequence of the coronavirus N protein is shown as SEQ ID NO: 1, and the following components:

the amino acid sequence is shown in SEQ ID NO. 2:

the invention also provides a method for expressing the N protein, which comprises cloning the N protein gene, expressing in the enterobacter coli, and purifying to obtain the corresponding N protein.

The invention also provides a method for preparing the monoclonal antibody with the specificity of the N protein, which comprises the steps of immunizing a mouse by taking the N protein as an immunogen, and preparing and obtaining the monoclonal antibody.

The invention firstly uses N protein to immunize a mouse, then spleen lymphocytes of the successfully immunized mouse are fused with myeloma cells, and a hybridoma cell strain of a specific secretion anti-N protein monoclonal antibody is obtained by screening positive clones, is used for producing a large amount of anti-N protein monoclonal antibodies, and is prepared to obtain the high-purity anti-N protein monoclonal antibody in a protein purification mode.

The invention particularly relates to a monoclonal antibody, wherein the heavy chain variable region amino acid sequence of the monoclonal antibody is SEQ.ID.NO.9:

QIQLQQSGASLVRPGALVKLSCKASDSHYYSQSLHWVKQRPVQGLEWIGDIGSADNSTKYAPPFL GKAQITADTSSNTAYLQLSSLTSEDTAVYYCIRYASSGWGWGQGTTLTVSS。

the amino acid sequence of the light chain variable region sequence is SEQ.ID.NO.10:

DIVITQSPALMADSPGEKVTITCDYSEEISMSYLAWYQQSSGISPKPWIYTTAGLAPGVPARFSG SGSGTSYSLTITSAEAEDAATYYCSSASSLPCRFGAGTKLELK。

the invention also provides an application of the anti-N protein monoclonal antibody, and the application is to prepare an ELISA reagent kit.

The invention relates to an application of an anti-N protein monoclonal antibody, in particular to an application of the anti-N protein monoclonal antibody in immunological detection, in particular to the detection of N protein, thereby identifying whether coronavirus exists or not.

The invention has the beneficial effects that:

the N protein is an important marker protein of the coronavirus, and the detection of the N protein can realize the detection of the coronavirus. The anti-N protein monoclonal antibody obtained by the invention has the characteristics of high specificity and mass production, and the titer of the obtained antibody is high. The anti-N protein monoclonal antibody obtained by the invention can be specifically combined with N protein, can be used for immunological detection of cells, has wide market prospect and has important clinical significance.

Drawings

FIG. 1 is a comparison between before and after the optimization of the N protein gene, wherein 1A is a diagram before the optimization and 1B is a diagram after the optimization.

FIG. 2 is a graph of antibody titer results; the dark column is antibody and the light column is control.

FIG. 3 shows the result of Western blot experiment of N protein with mAb monoclonal antibody. 1 is a self-made marker, 2-4 are lysates of blank host bacteria BL21(DE3), blank host bacteria BL21(DE3), blank plasmid lysates, and 5 is N protein.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Example 1 epitope optimization of N protein

According to the whole genome sequence already disclosed by 2019-nCoV, an optimized N protein epitope amino acid sequence is designed aiming at the N protein amino acid sequence peptide. The sequence is analyzed by 5 amino acid sequences of different strains such as Chinese strain, Italian strain and the like through Clustal W (Version1.81) software multi-sequence alignment, and the immunogenicity, the hydrophilicity, the surface possibility and the related secondary structure of the strain are analyzed according to DNAstar. A three-dimensional structure of the protein is constructed through Modeller homology modeling, the surface possibility of the immunogen polypeptide is further analyzed, an antibody binding epitope peptide is simulated through MOE software, interacting amino acids are analyzed, an N protein sequence with better immunogenicity is obtained through screening, and in comparison between a graph shown in figure 1A and a graph shown in figure 1B, the optimized codon sequence shown in figure 1B enables the GC content in the N protein sequence to be 50.40, so that the N protein sequence is more suitable for protein expression.

The nucleotide sequence is shown as SEQ ID NO: 1, and the following components:

the amino acid sequence is shown in SEQ ID NO. 2:

EXAMPLE 2 expression of N protein

Designing specific primers according to the optimized N protein nucleotide sequence, and respectively adding protective bases and restriction endonucleases into the 5' ends of the primers, wherein the primer sequences are as follows:

F:5'-CGCGGATCCATGAAAGACCTGTCTCCGCG-3'(SEQ.ID.NO.3)：

R:5'-CCGGAATTCGTCGTCCAGTTTGATAGCAC-3'(SEQ.ID.NO.4)：

the optimized N protein gene synthesized by the whole gene is used as a template (synthesized in Shanghai), and PCR amplification is carried out by using the specific primer.

The PCR reaction program is: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 10sec, annealing at 55 ℃ for 35sec, extension at 67 ℃ for 1min for 10s, 35 cycles; extension at 68 deg.C for 7min, and storage at 4 deg.C.

And (3) PCR reaction system: 2 XPCR Buffer 12.5. mu.L, 2mM dNTPs 5. mu.L, 10mM R Primer 0.75. mu.L, 10mM F Primer 0.75. mu.L, N DNA 1. mu.L, dH₂O 5 μL。

The amplified product was double-digested with restriction enzymes BamHI and EcoRI and vector pColdI, respectively, in a 9.5. mu.L PCR product or vector, 0.5. mu.L BamHI, 0.5. mu.L EcoRI, 8.5. mu.L dH₂And O. And reacting for 1h at 37 ℃. Carrying out the enzyme digestion1% agarose gel electrophoresis, and recovery and purification by using an agarose gel DNA purification kit from Takara.

The enzyme digestion product N gene and pColdI are connected by adopting a Ligation Kit DNA Ligation Kit (Mighty Mix) of Takara company, the mol ratio of the prepared recombinant prokaryotic expression vector pCold-N.N and pColdI is about 3: 1.16 ℃, the connection is carried out for 1 hour, the Ligation product is transformed into an escherichia coli competent cell DH5 α in a heat shock transformation mode, overnight culture is carried out at 37 ℃, and a positive plasmid with positive colony PCR and enzyme digestion identification is selected for sequencing identification, and the sequence is consistent with SEQID NO: 1 after identification.

The correctly sequenced plasmid pCold-N was transformed into an expression form of E.coli competent cells BL21 by means of heat shock transformation (DE 3). And (5) determining positive bacteria by colony PCR, namely expression bacteria containing recombinant expression plasmid pCold-NS 3. Inoculating an expression bacterium containing a recombinant expression plasmid pCold-NS3 into an autoinduction expression medium to induce expression, wherein the autoinduction medium is Overnight express (TM) Instant LB Media, and the step of inducing expression comprises the following steps: a small amount of the bacterial liquid was inoculated into 3mL of LB liquid medium containing Amp and cultured overnight at 37 ℃. Adding the overnight cultured bacterial liquid into an Amp-containing self-induction culture medium according to the proportion of 1:100, and inducing for 22h at the temperature of 17 ℃. The expression product is purified by recombinant protein through a QIAGEN His-tag protein purification kit, and then SDS-PAGE analysis is carried out, and the result shows that the protein accounts for more than 51 percent of the total expression protein amount by the method provided by the invention, and the expression amount is extremely obvious. The concentration of the protein after the preparation and purification is 0.5mg/L for standby.

EXAMPLE 3 preparation of monoclonal antibodies

Balb-c mice were 15 female, 8 weeks old, and had a body mass (20. + -.2 g) provided by the center of laboratory animals at Yangzhou university. The groups were randomized into 3 groups of 5 individuals. The N protein obtained in example 2 is used as immunogen, after fully emulsifying with equivalent Freund's complete adjuvant, the antigen is injected into mice at multiple points at subcutaneous sites according to 70 ug/mouse, 2 weeks later, 2 nd immunization is carried out with Freund's incomplete adjuvant emulsion antigen, 3 times of immunization are followed by detection of ELISA titer > 1: after 1 ten thousand, 50g of antigen is used for abdominal cavity impact immunization, and fusion is carried out after 3-4 days.

Taking a spleen of a mouse aseptically, preparing splenocytes, and fusing the splenocytes with myeloma SP2/O cells in a logarithmic growth period by using PEG4000 with the volume fraction of 50 percent; suspending the fused cells by using HAT culture medium, culturing in a 96-well plate in a CO2 culture box with the temperature of 37 ℃ and the volume fraction of 5%, replacing 1/2HAT culture medium by HT after 5 days, replacing the HT culture medium by using HT after 7-10 days, regularly observing, screening positive clones by adopting an indirect ELISA method 2 days after 2-3 times of liquid replacement, and continuing subcloning for 2 times by adopting a limiting dilution method. An ELISA plate is coated by N protein, and after 3 times of subcloning, a positive monoclonal cell line with good morphology, vigorous growth and stable reaction is obtained and named as N-32 after ELISA result screening. The ascites is purified by salting out with saturated ammonium sulfate. And (4) carrying out protein quantification by using an ultraviolet spectrophotometer. Purification gave mAb at a concentration of 2.0 g/L.

Example 4 identification of mAbs

(1) Subclass identification: goat anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM were subjected to immunodiffusion with 10-fold concentrated hybridoma cell culture supernatant, and formation of a precipitation line was observed. The mAb is detected to be IgG2a subtype by immune double-diffusion detection.

(2) Determination of potency and relative affinity:

and (3) measuring the titer:

diluting the N protein to 1 mu g/mL by using 0.05mol/L carbonate buffer solution with pH9.6, coating an enzyme label plate, carrying out 100 mu L/hole and standing overnight at 4 ℃; the monoclonal antibodies were mixed at a ratio of 1:10 with a sample diluent (phosphate buffer containing 0.5% bovine serum albumin)³、1:10⁴、1：10⁵、1：10⁶、1：10⁷、1：10⁸、 1：10⁹、1：10¹⁰Diluting, 100 mu L/hole, and incubating for 1h at 37 ℃; taking out the ELISA plate, washing for 3 times by TBS-T, patting the ELISA plate dry, adding 100 μ L of HRP-labeled goat anti-mouse secondary antibody diluted by 1:5000 into each hole, and incubating for 30min at 37 ℃; after TBS-T washing for 5 times, adding 100 μ L of TMB substrate display solution into each well, developing in dark at 37 deg.C for 10-15min, adding 50 μ L of stop solution to stop reaction, reading light absorption value at 450nm wavelength of microplate reader, and the result is shown in FIG. 2, where the titer of the purified monoclonal antibody reaches 8.6 × 10^-9。

And (3) affinity identification: the different concentrations of the mAbs to be tested are plotted on the abscissa and the corresponding A values are plotted on the ordinate to obtain a curve, the A value of the upper plateau of the curve is 100%, and the concentration of the mAb at the point where the A value is 50% represents the relative affinity of the mAb. The results showed that the relative affinity of the mAb determined by indirect ELISA was 1.835X 10^-10mol/L, shows better affinity.

(3) And (3) specific identification: the binding activity of the mAb to the N protein was determined by Western blot: carrying out SDS-PAGE on the purified N protein, transferring the gel protein to a nitrocellulose membrane in a charged manner, sequentially reacting with mAb and HRP-goat anti-mouse IgG, then developing the gel with DAB, carrying out cross-reactivity detection on the mAb and corresponding N protein antigens of HBV, HEV and HCV antigens, and adopting indirect ELISA.

Western blot identification as shown in FIG. 3 indicates that mAb can recognize a band with an Mr of about 20-30KD, indicating binding to the N protein of interest; and has no cross reaction with lysate of host bacterium BL21(DE3) and empty plasmid lysate. And the indirect ELISA method is used for determining that the mAb has no binding reaction with the corresponding N protein antigens of HBV, HEV and HCV antigens.

Example 5 identification of mAb sequences

The selected hybridoma cells of example 3 were collected, total RNA of the hybridoma cells was extracted using Trizol of Thermo, and mRNA was reverse-transcribed into cDNA according to the protocol of HiScript Q RT Supermix for qPCR (+ gDNA wiper) of Nanjing Novonza, and frozen at-20 ℃ for use. The reverse transcription system consisted of 5. mu.L of RNA (2500ng), 10. mu.L of 4 XgDNA, 10. mu.L of 5 Xsupermix II, and ddH2O to make up to 50. mu.L, with a total reaction volume of 50. mu.L.

Using cDNA as a template, searching the gene sequences of a murine heavy chain FR1 region and a hinge region through an NCBI database, designing a heavy chain PCR primer according to the sequences, wherein the upstream sequence is (SEQ. ID. NO. 5): GCGCTCGAGCAGKTCCAGCTGAAGCAGTC, the downstream sequence is (SEQ. ID. NO. 6): GCGACTAGTGCATTTGCATGGAGGACAG, respectively; similarly, the murine FR1 region of light chain and the gene sequence of constant region (NC000072.6) were searched by NCBI database, and light chain PCR primers were designed, with the upstream sequence (seq. id No. 7): GCGGAGCTCGATRTTGTGATGACCCARAC, the downstream sequence is (SEQ. ID. NO. 8): GCGTCTAGACTCATTCCTGTTGAAGCTCT, the light and heavy chain fragments of the antibody were obtained by PCR, respectively. The PCR was carried out according to the protocol of Phanta Max Super-Fidelity DNA Polymerase of Nanjing Nodezak company, and the PCR reaction system was: 25. mu.L of 2 XPphanta, 1. mu.L of dNTP, 4. mu.L of 10. mu.M primer pair, 4. mu.L of hybridoma cDNA, 1. mu.L of DNApolymerase, 15. mu.L of dd H2O, and a total reaction volume of 50. mu.L. The amplification conditions were: pre-denaturation at 94 deg.C for 3 min; denaturation at 94 ℃ for 30 s; annealing at 64 ℃ for 30 s; extension was 72 ℃ for 5 min. And (3) performing Gel recovery on the PCR product according to the instruction of an OMEGA Gel Extraction Kit of the OMEGA company, and performing sequencing analysis to obtain the heavy chain variable region amino acid sequence of the hybridoma cell as SEQ.ID.NO.7 and the light chain variable region amino acid sequence as SEQ.ID.NO. 8.

The heavy chain variable region amino acid sequence is SEQ.ID.NO.9:

QIQLQQSGASLVRPGALVKLSCKASDSHYYSQSLHWVKQRPVQGLEWIGDIGSADNSTKYAPPFL GKAQITADTSSNTAYLQLSSLTSEDTAVYYCIRYASSGWGWGQGTTLTVSS

it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

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Claims

1. An N protein, characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

2. An N protein-encoding gene characterized in that: the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

3. An anti-N protein monoclonal antibody characterized by: wherein the heavy chain variable region amino acid sequence of the antibody is SEQ.ID.NO: 9, the amino acid sequence of the light chain variable region sequence is SEQ.ID.NO: 10.

4. use of the anti-N protein monoclonal antibody of claim 3 in the preparation of a kit for detecting N protein.

5. A kit comprising the monoclonal antibody of claim 3.

6. Use of an anti-N protein monoclonal antibody according to claim 3 for the preparation of a kit for the detection of a novel coronavirus.

7. A novel coronavirus detection method is characterized in that: detection using the kit of claim 5.