CN113024641B - Novel recombinant S protein of coronavirus, preparation method and application thereof - Google Patents
Novel recombinant S protein of coronavirus, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a novel recombinant S protein of coronavirus, a preparation method and application thereof, wherein the amino acid sequence of the recombinant S protein is shown as SEQ ID NO. 1. IgG and IgM antibody activation epitope polypeptides are further screened and obtained based on a Corona S1 structural domain, and a recombinant S protein shown as SEQ ID NO. 1 is assembled by utilizing tandem polypeptides. The recombinant S protein is applied to the detection of novel coronavirus IgG and IgM antibodies, so that the false positive rate of novel coronavirus diagnosis can be effectively reduced, and the detection accuracy is improved.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a novel recombinant S protein of coronavirus, and a preparation method and application thereof.
Background
The novel coronavirus pneumonia is an acute infectious pneumonia, and the pathogen of the novel coronavirus is a novel coronavirus which is not found in human before, namely 2019 novel coronavirus. In 7 days 2 months in 2020, the national Weijian Commission decides to temporarily name the pneumonia infected by the novel coronavirus as the novel coronavirus pneumonia, which is called as the novel coronavirus pneumonia for short. On day 11 of 2 months, the World Health Organization (WHO) named its English name Corona Virus Disease 2019 (COVID-19). On day 22, 2 months, the national defense and health committee decided to revise the English name of the novel coronavirus pneumonia to COVID-19, which is consistent with the name of the world health organization, and the Chinese name remains unchanged. The initial symptoms of patients with the novel coronavirus pneumonia are mostly fever, hypodynamia and dry cough, and severe manifestations such as dyspnea gradually appear. The prognosis is good in most patients, and acute respiratory distress syndrome or septic shock may occur in some severe cases, even though death. At present, effective antiviral drugs aiming at pathogens are lacked, and isolation treatment and symptomatic support treatment are mainly used. Based on the current epidemiological investigation, the incubation period is 1 to 14 days, and is mostly 3 to 7 days. The infectious agents are mainly patients infected by the novel coronavirus and asymptomatic infected persons. The latent period is infectious, asymptomatic infectors can also become sources of infection, and the population is generally susceptible. After infection or inoculation with a new coronavirus vaccine, a certain immunity can be obtained, but the duration is not clear. Transmission via respiratory droplets and intimate contact is the primary route of transmission, and contact with viral contaminated items can also cause infection. There is a potential for transmission through aerosols when exposed to high concentrations of aerosols for extended periods of time in a relatively closed environment.
The previously known human-infecting coronaviruses are 6, namely HCoV-229E, HCoV-OC43, SARSr-CoV, HCoV-NL63, HCoV-HKU1 and MERSR-CoV. The novel coronavirus pneumonia is mainly caused by 2019 novel coronavirus (2019-nCoV) infection, belongs to a coronavirus of beta genus, and has obvious gene characteristics different from SARSr-CoV and MERSR-CoV. A Novel Coronavirus (Novel Server reactive resonance Coronavir 2, SARS-CoV-2) is a single-stranded positive-strand RNA virus and has extremely strong infectivity and lethality. The novel coronavirus is a spherical virus, mainly comprising 4 structural proteins, namely Spike Protein (S), Membrane glycoprotein (M), small envelope glycoprotein (E) and nucleocapsid Protein (N), wherein the three proteins are scattered on the surface of a virus capsid. The S protein and the N protein are the most important target proteins, the N protein is a high-immunogenicity phosphoprotein, and in the process of assembling the virosome, the N protein is combined with viral RNA to form a spiral nucleocapsid and is related to replication of a viral genome and regulation of a cell signal path, so that the N protein is often used as a coronavirus diagnosis and detection tool and is a core raw material of an immunological rapid diagnosis reagent. The S protein has the functions of binding virus and host cell membrane receptor and fusing membrane, and is the key target of host neutralizing antibody and vaccine design. The N protein is the most abundant protein in the coronavirus and has high conservation. The human or animal body has no obvious symptoms (generally 4-7 d) in the initial stage of infection of the novel coronavirus, but S protein and E protein on the surface of the virus can already stimulate humoral immunity of a host to secrete IgM antibodies, and IgG antibodies are mainly secreted after one week. Therefore, the membrane surface antigen is suitable for the novel coronavirus antibody serum detection.
Clinically, the novel coronavirus needs to be identified with other known viral pneumonia such as influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus, rhinovirus, human metapneumovirus, SARS coronavirus and the like, and with mycoplasma pneumoniae, chlamydia pneumonia, bacterial pneumonia and the like. In addition, SARS-CoV-2 is also identified from non-infectious diseases, such as vasculitis, dermatomyositis, and organized pneumonia. Antibody detection is the most convenient means for diagnosing the novel coronavirus besides PCR detection.
However, the accuracy of the detection kit for the antibody of the new coronavirus using a general antigen is not high, and a false positive detection result is easily generated.
Disclosure of Invention
Based on this, there is a need to provide a recombinant S protein for detecting a novel coronavirus with a low false positive rate by using a novel coronavirus antibody.
A recombinant S protein of a novel coronavirus has an amino acid sequence shown as SEQ ID NO. 1.
The novel coronavirus S protein comprises two functional domains, namely Corona S1 and Corona S2, wherein the Corona S2 functional domain is highly similar to other coronaviruses, and Corona S1 is a recognition region of a host ACE2 receptor and has low similarity with other coronaviruses. Therefore, IgG and IgM antibody activation epitope polypeptides are further screened based on the Corona S1 structural domain, and the recombinant S protein shown as SEQ ID NO. 1 is assembled by utilizing the tandem polypeptides. The recombinant S protein is applied to the detection of novel coronavirus IgG and IgM antibodies, so that the false positive rate of novel coronavirus diagnosis can be effectively reduced, and the detection accuracy is improved.
The invention also provides a nucleic acid capable of expressing a recombinant S protein as described above.
In one embodiment, the nucleotide sequence of the nucleic acid is set forth in SEQ ID NO 2.
The invention also provides a recombinant expression vector containing the nucleic acid.
The invention also provides a host cell, the genome of which incorporates a nucleic acid as described above.
The invention also provides a preparation method of the recombinant S protein, which comprises the following steps: culturing the host cell under proper conditions, collecting culture solution and/or lysate of the host cell, and then separating and purifying to obtain the recombinant S protein.
The invention also provides the application of the recombinant S protein, the nucleic acid, the recombinant expression vector or the host cell in preparing products for detecting the novel coronavirus antibody.
The present invention also provides a method for detecting a novel coronavirus antibody for non-disease diagnosis and treatment purposes, which detects the novel coronavirus antibody by specific binding of the antigen-antibody using the recombinant S protein as described above as an antigen.
In one embodiment, the preparation method comprises the following steps: and (3) incubating the recombinant S protein for one night by using 0.01-0.03M phosphate buffer solution according to the plate package of 0.1-1 microgram/hole, washing the plate, sealing, adding a sample to be tested for incubation, washing the plate again, incubating and combining by using a marked anti-antibody, and performing reaction color development after the anti-antibody reaction is finished.
The invention also provides a new coronary pneumonia detection kit, which comprises the recombinant S protein, the anti-antibody and a solid support;
the anti-antibody is an antibody against immunoglobulin of a test sample source species;
optionally, the recombinant S protein is conjugated to the solid support;
optionally, the solid support is a test tube, an EP tube, a multi-well plate, a microplate well, a bead, or a small disk;
optionally, the anti-antibody is conjugated to a signal substance.
Drawings
FIG. 1 is a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis result of the recombinant bacteria induced expression product in example 2;
FIG. 2 is a SDS-PAGE analysis result of an expression product of the recombinant bacterium in example 2 at 37 ℃;
FIG. 3 is a SDS-PAGE analysis result of an expression product of the recombinant bacterium of example 2 at 30 ℃;
FIG. 4 is a SDS-PAGE analysis result of the expression product of the recombinant bacterium in example 2 at 25 ℃;
FIG. 5 is a SDS-PAGE analysis result of an expression product of the recombinant bacterium in example 2 under a culture condition of 16 ℃;
FIG. 6 is a graph showing the results of capturing novel coronavirus IgG antibodies by immunoblotting in example 3;
FIG. 7 is a graph showing the results of capturing novel coronavirus IgM antibodies by immunoblotting in example 3;
FIG. 8 is a graph comparing ELISA results of recombinant S protein (soluble) in example 4 under different conditions.
Detailed Description
In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Interpretation of terms
"antigen" refers to all substances that induce the immune response of the body, i.e., substances that are specifically recognized and bound by antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, proliferate and differentiate to produce immune response products (sensitized lymphocytes or antibodies), and specifically bind to the corresponding products in vitro and in vivo. Thus, antigens have two basic properties, namely antigenicity and immunogenicity. Antigenicity refers to the ability of an antigen to specifically bind to the antibody or sensitized lymphocyte it induces. Immunogenicity refers to the property of eliciting an immune response, i.e., the ability of an antigen to stimulate specific immune cells, activate, proliferate, differentiate, and ultimately produce immune effector antibodies and sensitized lymphocytes.
"antibody" refers to a class of immunoglobulins that specifically bind to an antigen. Antibodies are classified by their reactive forms into lectins, sinkers, antitoxins, lysins, opsonins, neutralizing antibodies, complement-binding antibodies, and the like. The antibody production sources are classified into normal antibodies (natural antibodies), such as anti-A and anti-B antibodies in blood group ABO type, and immunological antibodies such as antimicrobial antibodies. The antigen is classified into xenogenous antibodies, heterophilic antibodies, allogenic antibodies and autoantibodies according to the source of the antigen. The agglutination state of the antigen reaction is classified into a complete antibody IgM and an incomplete antibody IgG. Antibodies are widely used in medical practice, and have certain effects in preventing, diagnosing and treating diseases. Clinically, the gamma globulin is used for preventing viral hepatitis, measles, rubella and the like, and the anti-Rh immunoglobulin is used internationally for preventing hemolysis caused by Rh blood group incompatibility. For diagnosis, rheumatoid factor is used for rheumatoid arthritis, antinuclear antibody (ANA) and anti-DNA antibody are used for systemic lupus erythematosus, and antisperm antibody is used for diagnosis of primary infertility. The treatment includes antitoxic treatment for toxin poisoning, and treatment for immunodeficiency diseases.
"vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40).
"host cell" means a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.
The amino acid sequence of the recombinant S protein of the novel coronavirus is shown as SEQ ID NO. 1.
The novel coronavirus S protein comprises two functional domains, namely Corona S1 and Corona S2, wherein the Corona S2 functional domain is highly similar to other coronaviruses, and Corona S1 is a recognition region of a host ACE2 receptor and has low similarity with other coronaviruses. Therefore, IgG and IgM antibody activation epitope polypeptides are further screened based on the Corona S1 structural domain, and the recombinant S protein shown as SEQ ID NO. 1 is assembled by utilizing the tandem polypeptides. The recombinant S protein is applied to the detection of novel coronavirus IgG and IgM antibodies, so that the false positive rate of novel coronavirus diagnosis can be effectively reduced, and the detection accuracy is improved.
The nucleic acid of an embodiment of the present invention, which is capable of expressing the recombinant S protein as described above.
In a specific example, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 2. It will be appreciated that due to the degeneracy of the codons, the nucleic acid sequences capable of expressing the same protein have a variety of forms, the above being codon optimized nucleic acid sequences, but are not limited thereto.
The recombinant expression vector of an embodiment of the present invention contains the nucleic acid as described above.
In a specific example, the recombinant expression vector is constructed based on pET-30a (+), but is not limited thereto. It will be appreciated that the vector may also contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, etc., and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).
The host cell of an embodiment of the present invention has a genome into which a nucleic acid as described above is incorporated.
In a specific example, the host cell is Escherichia coli, Bacillus subtilis, yeast cell, Aspergillus, or the like. In a specific example, the host cell is preferably a prokaryotic cell, preferably escherichia coli, and more preferably escherichia coli BL21 strain.
The preparation method of the recombinant S protein of one embodiment of the invention comprises the following steps: culturing the host cell under proper conditions, collecting the culture solution and/or the host cell lysate, and then separating and purifying to obtain the recombinant S protein.
In one specific example, the preparation method comprises the following steps: inoculating the recombinant Escherichia coli into a liquid culture medium, culturing at 37 ℃ to a logarithmic phase, adding an IPTG inducer for continuous culture, then collecting thalli, cracking the thalli and centrifugally collecting supernatant. Preferably, 0.08 mM-0.12 mM IPTG is used for inducing for more than 4h at 24-26 ℃.
In the method for detecting a novel coronavirus antibody according to an embodiment of the present invention, the novel coronavirus antibody is detected by specific binding of the antigen-antibody using the recombinant S protein as an antigen. It is understood that the specific method can be selected from immunoblotting, enzyme-linked immunosorbent assay, immunochromatography, and the like, but is not limited thereto, and any method using the principle of specific binding of antigen and antibody may be used.
Immunoblotting (also known as Western blotting) is a method for detecting a protein in a complex sample based on the specific binding of an antigen and an antibody. The method is a new immune biochemical technology developed on the basis of gel electrophoresis and solid-phase immunoassay. Immunoblotting has become a common technique for protein analysis due to the high resolution of SDS-PAGE and the high specificity and sensitivity of solid phase immunoassays. Immunoblotting is commonly used to identify certain proteins and enables qualitative and semi-quantitative analysis of proteins. Combining with chemiluminescence detection, the expression quantity difference of the same protein of a plurality of samples can be simultaneously compared.
Enzyme linked immunosorbent assay (ELISA), which is a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid phase carrier such as polystyrene and immunoreaction is carried out by utilizing specific binding of antigen and antibody. The basic principle of this method is: binding antigen or antibody to the surface of certain solid phase carrier and maintaining its immunological activity; ② the antigen or antibody is connected with certain enzyme to form enzyme-labeled antigen or antibody, and the enzyme-labeled antigen or antibody not only retains its immunological activity, but also retains the activity of enzyme. In the measurement, the specimen to be tested (the antibody or antigen to be measured therein) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid carrier in a different step. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the enzyme quantity bound on the solid phase carrier is in a certain proportion to the quantity of the detected substance in the specimen. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to be changed into a colored product, and the amount of the product is directly related to the amount of the detected substance in the sample, so that qualitative or quantitative analysis can be carried out according to the existence of the color reaction. Because the catalytic efficiency of the enzyme is very high, the reaction effect can be greatly amplified, so that the determination method achieves very high sensitivity.
The principle of immunochromatography (immunochromatography) is that a specific antibody is firstly fixed in a certain zone of a nitrocellulose membrane, after one end of the dried nitrocellulose membrane is immersed in a sample (urine or serum), the sample moves forwards along the membrane due to capillary action, when the sample moves to a region where the antibody is fixed, a corresponding antigen in the sample is specifically bound with the antibody, and the region can show a certain color if the region is stained by immune colloidal gold or immune enzyme, so that specific immunodiagnosis is realized. For example, the colloidal gold test paper is prepared by fixing colloidal gold-labeled biomacromolecules on a PVC test paper, reserving a sample adding hole, and arranging a detection line and a quality control line. And after the sample is added into the sample adding hole, the liquid medium is dripped into the sample adding hole, and chromatography is carried out on the test paper. And judging the detection result according to the immunoreaction occurrence result on the test paper, namely whether a red strip appears at the position of the colloidal gold.
In one specific example, the detection method comprises the following steps: and (3) incubating the recombinant S protein for overnight by using a buffer solution according to the condition that the recombinant S protein is coated in a range of 0.1 mu g/hole to 1 mu g/hole, sealing after washing the plate, adding a sample for incubation, washing the plate again, incubating and combining by using a marked anti-antibody, and performing reaction and color development by using a substrate solution after the anti-antibody reaction is finished. Preferably, the buffer is a phosphate buffer, more preferably a phosphate buffer with pH of 7.20.01M-0.03M, and the coating concentration is 0.4. mu.g-0.6. mu.g/well. For example, the recombinant S protein is coated on a plate by 0.5 mu g/hole, 100 mu L/hole and incubated overnight at 4 ℃, after the plate is washed, 5% skimmed milk powder is used for sealing, rabbit anti-novel coronavirus S protein polyclonal IgG antibody is added for incubation for 1 hour, after the plate is washed again, goat anti-rabbit IgG antibody marked by HRP is used as a secondary antibody for incubation and combination for 45min, after the secondary antibody reaction is finished, TMB substrate solution is used for reaction and color development, and the OD450nm value is measured.
In one specific example, the detection method comprises the following steps: and (3) carrying out SDS-PAGE electrophoresis on the recombinant S protein sample, carrying out membrane transfer and antibody incubation after the electrophoresis is finished, wherein the primary antibody is incubated with the sample, and the secondary antibody is developed with the anti-human monoclonal antibody by using a chemiluminescence kit. For example, the recombinant S protein is subjected to SDS-PAGE electrophoresis in a loading amount of 10. mu.g, and after the electrophoresis is finished, membrane transfer and antibody incubation are performed, wherein a serum sample for primary antibody and a monoclonal antibody for secondary antibody are used for a mouse anti-human IgG monoclonal antibody or a mouse anti-human IgM monoclonal antibody, and then are developed by a hypersensitivity ECL chemiluminescence kit, and photographs are taken on a Brillo chemiluminescence imaging system.
The new coronary pneumonia detection kit of one embodiment of the present invention contains the recombinant S protein, the anti-antibody and the solid support as described above. It is understood that an anti-antibody is an antibody against an immunoglobulin of the species from which the sample is derived.
Alternatively, the recombinant S protein is conjugated to a solid support. Optionally, the solid support is a test tube, an EP tube, a multi-well plate, a microplate well, a bead, or a small disk. Optionally, the anti-antibody is conjugated to a signal substance. Optionally, the detection kit further comprises one or more of phosphate buffer, skim milk powder and 3,3',5,5' -tetramethylbenzidine.
Embodiments of the present invention will be described in detail below with reference to specific examples.
Example 1 preparation of recombinant bacteria
After synthesizing the codon optimized recombinant S protein gene (SEQ ID NO:2), the restriction enzyme double digestion of EcoR I and Xho I is carried out simultaneously with a prokaryotic expression vector pET-30a (+), nucleic acid fragments are respectively recovered, and then T4 DNA ligase is used for connection overnight.
The ligation product is heat shock transformed into BL21(DE3) infected cells, and after antibiotic pressure screening is carried out by using a 50 mu g/mL kanamycin LB culture plate, colony PCR identification and nucleic acid sequencing analysis are carried out, a recombinant bacterium is obtained, and the bacterium also comprises a recombinant vector.
Example 2 expression of recombinant S protein
By varying the induction temperature (T), induction time (T) and IPTG concentration, the optimal induction conditions to obtain soluble expression of recombinant S protein were determined.
Picking single colony of the recombinant bacteria to 5mL LB liquid culture medium containing 50 ug/mL kanamycin, after overnight culture, inoculating 1% of the single colony to 100mL LB liquid culture medium containing 50 ug/mL kanamycin, culturing at 37 ℃ until logarithmic phase, adding IPTG inducer with different concentrations, continuing culturing for 4h, and collecting the bacteria. PBS was added to the cells, and after washing twice in succession, 10mL of PBS containing 1% Triton X-100 was added, the cells were sonicated on ice, centrifuged at 12000rpm for 20min, and the supernatant Q1 was collected. The remaining precipitate was added with loading buffer (100mM NaH)2PO410mM Tris, 8M urea, 10mM 2-ME, pH8.0), followed by centrifugation at 12000rpm for 30min, and collecting the supernatant Q2. The supernatant samples Q1 and Q2 were both detected by SDS-PAGE analysis, and the results are shown in FIG. 1. Lane M is Protein Marker, lane 1 is the whole bacterial Protein of wild expression strain, lane 2 is the whole bacterial Protein of recombinant strain induced at 0.1mM IPTG 37 ℃ for 4h, lane 3 is the whole bacterial Protein of recombinant strain induced at 0.5mM IPTG 37 ℃ for 4h, lane 4 is the whole bacterial Protein of recombinant strain induced at 1mM IPTG 37 ℃ for 4h, lane 5 is the whole bacterial Protein of recombinant strain induced at 0.1mM IThe supernatant from the lysis after 4h induction at 37 ℃ in PTG, lane 6 is the pellet of the recombinant bacteria after 4h induction at 37 ℃ in 0.1mM IPTG. The result shows that the recombinant bacteria can effectively express the target protein through induction, and the size of the recombinant bacteria is about 30 KDa.
Similarly, the expression products were analyzed by SDS-PAGE under 0.5mM IPTG induction at 30 ℃ for different culture times, and the results are shown in FIG. 2. Lane M is Protein Marker, lane 1 is 30 ℃ 2h induction bacteria solution, lane 2 is 30 ℃ 2h lysis supernatant, lane 3 is 30 ℃ 2h lysis precipitate, lane 4 is 30 ℃ 4h before induction, lane 5 is 30 ℃ 4h lysis supernatant, lane 6 is 30 ℃ 4h lysis precipitate, lane 7 is 30 ℃ 6h induction bacteria solution, lane 8 is 30 ℃ 6h lysis supernatant, lane 9 is 30 ℃ 6h lysis precipitate. The results show that the recombinant bacteria can not effectively express the target protein under the culture condition of 30 ℃.
Similarly, expression products induced by IPTG at different concentrations were subjected to SDS-PAGE analysis after 4h induction at 25 ℃ as shown in FIG. 3. Lane M is Protein Marker, lane 1 is pre-induced, lane 2 is induced with 0.1mM IPTG 25 ℃, lane 3 is induced with 0.5mM IPTG 25 ℃, lane 4 is induced with 1mM IPTG 25 ℃, lane 5 is lysed supernatant with 0.1mM IPTG 25 ℃ and lane 6 is lysed pellet with 0.1mM IPTG 25 ℃. The results show that the recombinant bacteria can not effectively express the target protein under the culture condition of 25 ℃.
Similarly, expression products induced by IPTG at different concentrations were subjected to SDS-PAGE analysis after 4h induction at 16 ℃ as shown in FIG. 4. Lane M is Protein Marker, lane 1 is pre-induced, lane 2 is induced with 0.1mM IPTG 16 ℃, lane 3 is induced with 0.5mM IPTG 16 ℃, lane 4 is induced with 1mM IPTG 16 ℃, lane 5 is lysed supernatant with 0.1mM IPTG 16 ℃, and lane 6 is lysed pellet with 0.1mM IPTG 16 ℃. The results show that the recombinant bacteria can not effectively express the target protein under the culture condition of 16 ℃.
And (3) separating and purifying the target protein of the supernatant Q1 by using a Ni-Sepharose affinity chromatography column, collecting eluent to obtain the soluble protein of the recombinant S protein, wherein the concentration of the purified target protein is 0.14 mu g/mu L. The results are shown in FIG. 5, lanes 1-2 are all soluble proteins of the purified recombinant S Protein, and lane M is Protein Marker.
Example 3 immunoblotting
The recombinant S protein (soluble), the recombinant S protein (inclusion body purified) and the N protein were subjected to WB analysis, and the loading amounts were 2. mu.g, 10. mu.g, 9.4. mu.g, serum dilution factor 1: 300, after SDS-PAGE electrophoresis, membrane transfer and antibody incubation were performed, wherein the primary antibody was positive serum and the secondary antibody was either a mouse anti-human IgG monoclonal antibody (St. Cruis Biotechnology, cat # sc-69786) or a mouse anti-human IgM monoclonal antibody (St. Cruis Biotechnology, cat # sc-376317), developed with a hypersensitivity ECL chemiluminescence kit, and photographed in a Berle chemiluminescence imaging system. The results are shown in FIGS. 6 and 7, in lane 1, protein N, lane 2, recombinant S protein (inclusion body purified), lane 3, recombinant S protein (soluble), lane M, pre-stained rainbow protein Marker, 3 bands of both protein-incubated anti-IgG secondary antibodies, and recombinant S protein (soluble) and recombinant S protein (inclusion body purified), anti-IgM secondary antibodies. As can be seen from FIGS. 6 and 7, the recombinant S protein can effectively capture novel coronavirus IgG and IgM antibodies to generate antigen-antibody immunoreaction.
Example 4 enzyme-linked immunosorbent assay (ELISA)
In order to further optimize the capture and binding of the recombinant S protein to the antibody by ELISA detection means, coating solutions with 4 different pH values, such as 0.05M acetate buffer (pH2.0), 0.05M citrate buffer (pH5.0), 0.02M phosphate buffer (pH7.2), and 0.05M carbonate buffer (pH9.6), were prepared in this example.
The recombinant S protein was incubated overnight at 4 ℃ in 3 replicate wells with the 4 buffers described above, at 0.1. mu.g/well, 0.2. mu.g/well, 0.5. mu.g/well, 1. mu.g/well, and 100. mu.L/well, respectively. After washing, the plate was sealed with 5% skimmed milk powder, and then a rabbit anti-novel coronavirus S protein polyclonal IgG antibody (prepared by King Share Biotechnology Ltd.) was added and incubated for 1 hour, and after washing, the plate was incubated and bound for 45min with a goat anti-rabbit IgG antibody labeled with HRP as a secondary antibody. After the secondary antibody reaction was completed, the reaction was developed using a TMB substrate solution, and the OD450nm value was measured.
The data of the ELISA detection results of the recombinant S protein (soluble) are shown in Table 1 and FIG. 8 (data results are shown as sample well OD 450/blank well OD450), and the data of the ELISA detection results of the recombinant S protein (inclusion body purified) are shown in Table 2. The results show that the optimal coating solution is 0.02M phosphate buffer (pH7.2), and the antigen coating concentration is 0.5. mu.g, the recombinant S protein capture ELISA has higher sensitivity and repeatability.
TABLE 1
| 0.1μg | 0.2μg | 0.5μg | 1μg | |
| 0.05M acetate buffer (pH2.0) | 1.647798742 | 2.937106918 | 2.289308176 | 2.566037736 |
| 0.05M lemon buffer (pH5.0) | 2.343949045 | 4.47133758 | 5.025477707 | 4.375796178 |
| 0.02M phosphate buffer (pH7.2) | 2.992424242 | 6.575757576 | 7.007575758 | 6.689393939 |
| 0.05M carbonate buffer (pH9.6) | 2.695121951 | 4.725609756 | 4.432926829 | 3.93902439 |
TABLE 2
| 0.1μg | 0.2μg | 0.5μg | 1μg | |
| 0.05M acetate buffer (pH2.0) | 2.194968553 | 2.094339623 | 2.371069182 | 1.364779874 |
| 0.05M lemon buffer (pH5.0) | 4.484076433 | 4.49044586 | 4.515923567 | 2.719745223 |
| 0.02M phosphate buffer (pH7.2) | 8.96969697 | 8.613636364 | 8.204545455 | 5.46969697 |
| 0.05M carbonate buffer (pH9.6) | 5.024390244 | 5.841463415 | 6.018292683 | 6.512195122 |
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> institute of animal health of academy of agricultural sciences of Guangdong province
<120> novel recombinant S protein of coronavirus, preparation method and application thereof
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<211> 223
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Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Ala
1 5 10 15
Ala Tyr Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu
20 25 30
His Arg Ser Tyr Leu Thr Pro Gly Ala Ala Tyr Tyr Tyr Val Gly Tyr
35 40 45
Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Ala Ala Tyr Thr Arg Phe
50 55 60
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Ala Ala Tyr Asn
65 70 75 80
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
85 90 95
Ala Ala Tyr Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys
100 105 110
Tyr Phe Pro Lys Lys Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
115 120 125
Val Gly Tyr Gln Pro Lys Lys Thr Glu Ile Tyr Gln Ala Gly Ser Thr
130 135 140
Pro Cys Asn Gly Val Glu Gly Lys Lys Cys Gly Pro Lys Lys Ser Thr
145 150 155 160
Asn Leu Val Lys Asn Lys Cys Val Asn Lys Lys Tyr Val Gly Tyr Leu
165 170 175
Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Lys Lys Val Gln Pro
180 185 190
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Lys Lys Ala
195 200 205
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu
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<213> Artificial Sequence (Artificial Sequence)
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ggttacttca aaatctactc taaacacacc ccgatcaacc tggttgctgc ttacatcggt 60
atcaacatca cccgtttcca gaccctgctg gctctgcacc gttcttacct gaccccgggt 120
gctgcttact actacgttgg ttacctgcag ccgcgtacct tcctgctgaa atacgctgct 180
tacacccgtt tcgcttctgt ttacgcttgg aaccgtaaac gtatctctgc tgcttacaac 240
tacctgtacc gtctgttccg taaatctaac ctgaaaccgt tcgaacgtgc tgcttacggt 300
tctaccccgt gcaacggtgt tgaaggtttc aactgctact tcccgaaaaa actgcagtct 360
tacggtttcc agccgaccaa cggtgttggt taccagccga aaaaaaccga aatctaccag 420
gctggttcta ccccgtgcaa cggtgttgaa ggtaaaaaat gcggtccgaa aaaatctacc 480
aacctggtta aaaacaaatg cgttaacaaa aaatacgttg gttacctgca gccgcgtacc 540
ttcctgctga aatacaacga aaaaaaagtt cagccgaccg aatctatcgt tcgtttcccg 600
aacatcacca acctgaaaaa agctgttgac tgcgctctgg acccgctgtc tgaaaccaaa 660
tgcaccctg 669
Claims (13)
1. A novel recombinant S protein of coronavirus is characterized in that the amino acid sequence of the recombinant S protein is shown as SEQ ID NO. 1.
2. A nucleic acid capable of expressing the recombinant S protein of claim 1.
3. The nucleic acid of claim 2, wherein the nucleotide sequence of the nucleic acid is set forth in SEQ ID NO 2.
4. A recombinant expression vector comprising the nucleic acid of claim 2 or 3.
5. A host cell having incorporated into its genome the nucleic acid of claim 2 or 3.
6. A method of producing the recombinant S protein of claim 1, comprising the steps of: culturing the host cell of claim 5 under suitable conditions, collecting the culture fluid and/or the lysate of the host cell, and then separating and purifying to obtain the recombinant S protein.
7. Use of the recombinant S protein of claim 1, the nucleic acid of any one of claims 2 to 3, the recombinant expression vector of claim 4 or the host cell of claim 5 for the preparation of a product for the detection of novel coronavirus antibodies.
8. A method for detecting a novel coronavirus antibody for non-disease diagnosis and treatment, which comprises detecting the novel coronavirus antibody by specific binding of an antigen-antibody using the recombinant S protein of claim 1 as an antigen.
9. The detection method according to claim 8, characterized by comprising the steps of: and (3) incubating the recombinant S protein for one night by using 0.01-0.03M phosphate buffer solution according to the plate package of 0.1-1 microgram/hole, washing the plate, sealing, adding a sample to be tested for incubation, washing the plate again, incubating and combining by using a marked anti-antibody, and performing reaction color development after the anti-antibody reaction is finished.
10. A new coronary pneumonia detection kit, comprising the recombinant S protein of claim 1, an anti-antibody, and a solid support;
the anti-antibody is an antibody against immunoglobulin of the species from which the sample is derived.
11. The neocoronary pneumonia detection kit of claim 10, wherein the recombinant S protein is conjugated to the solid support.
12. The assay kit of claim 10, wherein the solid support is a test tube, an EP tube, a multi-well plate, a microplate well, a bead, or a small disk.
13. The neocoronary pneumonia detection kit of claim 10 wherein the anti-antibody is conjugated to a signal substance.
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