CN113480619B - Polypeptide and application thereof in novel coronavirus detection - Google Patents

Polypeptide and application thereof in novel coronavirus detection Download PDF

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CN113480619B
CN113480619B CN202110990545.1A CN202110990545A CN113480619B CN 113480619 B CN113480619 B CN 113480619B CN 202110990545 A CN202110990545 A CN 202110990545A CN 113480619 B CN113480619 B CN 113480619B
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CN113480619A (en
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林军
刘滔滔
潘运宝
钱纯亘
汪大明
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Shenzhen Yhlo Biotech Co Ltd
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Abstract

The invention relates to the biomedical field, in particular to a polypeptide and application thereof in novel coronavirus detection. The polypeptide comprises the following parts: S-Linker-N-avi-tag. The S protein and the N protein are connected in series through the optimized rigid linker sequence, so that the two proteins have relatively independent space conformations, a plurality of dominant epitopes are added, and the sensitivity and the signal value are improved to a great extent; in addition, the fusion protein is introduced into the Avi-tag, so that the recombinant protein can be immobilized through the immobilized site, and the influence of steric hindrance brought by the coating process is reduced. Therefore, the polypeptide can achieve high sensitivity and specificity, and is not easy to miss detection.

Description

Polypeptide and application thereof in novel coronavirus detection
Technical Field
The invention relates to the biomedical field, in particular to a polypeptide and application thereof in novel coronavirus detection.
Background
2019, is designated as "2019-nCoV" by the world health organization, and is designated as "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" by the International Commission on the classification of viruses. The pneumonia patients infected with the new coronavirus take fever, hypodynamia, dry cough and the like as main symptoms, a few patients are accompanied with nasal obstruction, nasal discharge, diarrhea and other respiratory tract and digestive tract diseases, the serious patients can have dyspnea symptoms after 1 week, serious patients rapidly develop into acute respiratory distress syndrome, septic shock and the like, the life health of people is seriously threatened, and the global economy is greatly lost. The new coronavirus has strong transmissibility, high mortality and strong infectivity under asymptomatic condition, the latency period is as long as 14 days, and the high-quality diagnostic reagent is helpful for early finding infected people and cutting off the transmission path in time.
SARS-CoV-2 is a single positive strand RNA virus with envelope structure, belonging to beta coronavirus, which is elliptical or circular and has a diameter of 60-140nm. The Envelope is composed of Spike Protein (S), membrane Protein (M), envelope Protein (E), nucleocapsid Protein (N), wherein S1 and S2 are composed of two subunits. The S protein and the N protein have strong antigenicity and play an important role in inducing host immune response and pathogenesis. The S protein is the key of the invasion of human cells by the new coronavirus, and the specific binding is generated by the Receptor Binding Domain (RBD) on the S protein and the angiotensin converting enzyme 2 (ACE 2) of the host cells, so that the virus is mediated to enter the host cells. N protein and viral genome RNA are intertwined to form a viral nucleocapsid, which plays an important role in the synthesis process of viral RNA. The amino acid sequence of the N protein is relatively conserved, the ratio of the N protein to the structural protein is large, and the organism can produce high-level antibodies against the N protein in early infection. Therefore, the diagnostic reagents for the novel crown antibody mainly use spike protein (S) and nucleocapsid protein (N).
At present, the detection of SARS-CoV-2 is mainly based on PCR-based virus nucleic acid detection, and the exponential increase of the target gene is realized by PCR, so that the fluorescent signal of the target gene is collected to confirm that the sample contains virus nucleic acid. The nucleic acid detection method has higher specificity and sensitivity, and is used as a national gold standard for confirming whether infection exists or not. However, this detection method still has significant drawbacks, such as: the method has the advantages of high technical requirements, high equipment requirements (fluorescent PCR is needed), special treatment of specimens, easy occurrence of false negative, missed detection, and time-consuming and popularization-unfriendly early screening in communities, primary hospitals, customs and the like. It is therefore an urgent need to develop a new coronavirus diagnostic agent that is more early, accurate, rapid and effective.
After the organism is infected with pathogens, corresponding antigen components can be detected in vivo, particularly when the virus load is higher in the acute infection period, positive cases can be detected rapidly through antigen detection, and suspected patients can be diagnosed rapidly in an assisted manner and key people can be examined in an assisted manner. But the trusted material required by antigen detection has high sensitivity, high specificity, long antibody preparation period, interference of cross reaction with other SARS-CoV N proteins, and inferior sensitivity as compared with nucleic acid detection reagent, so that the method can be used as a supplement of the existing detection method. Pathogen invades the body, igM antibodies are the earliest produced antibodies in the human immune system, and specific IgM in serum increases, suggesting a recent infection. IgG antibodies are produced later than IgM, and are an indicator of past infection. Examination of IgM and IgG antibodies in the serum of suspected patients helps to diagnose SARS-CoV-2 infection, so serum antibody detection is very important evidence for diagnosing SARS-CoV-2 infection in patients with typical clinical characteristics.
Disclosure of Invention
The invention relates to a polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 1.
According to a second aspect of the invention, it relates to a nucleic acid encoding a polypeptide as described above.
According to a third aspect of the invention, it relates to a vector comprising a nucleic acid as described above.
According to a fourth aspect of the invention, it relates to a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.
According to a fifth aspect of the present invention, there is provided a novel coronatine detection kit comprising a polypeptide as described above, an anti-antibody and a solid support;
the anti-antibody is an antibody against the species immunoglobulin from which the sample is derived;
the AviTag tag in the polypeptide is pre-biotinylated and conjugated to the solid support via the biotinylated AviTag.
According to a seventh aspect of the present invention, there is provided the use of a polypeptide as described above in the preparation of a SARS-CoV-2 antibody detection test strip or kit.
The beneficial effects of the invention are as follows:
the S protein and the N protein are connected in series through the optimized rigid linker sequence, so that the two proteins have relatively independent space conformations, a plurality of dominant epitopes are added, and the sensitivity and the signal value are improved to a great extent; in addition, the fusion protein is introduced into the Avi-tag, so that the recombinant protein can be immobilized through the immobilized site, and the influence of steric hindrance brought by the coating process is reduced. Therefore, the polypeptide can achieve high sensitivity and specificity, and is not easy to miss detection.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a polypeptide according to one embodiment of the present invention;
FIG. 2 is a vector map of pCMV3L in one embodiment of the present invention;
FIG. 3 is a graph showing the result of electrophoresis of SARS-CoV-2-S+N recombinant antigen prepared in one embodiment of the present invention.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The term "polypeptide" or "protein" refers to a molecule comprising at least two amino acid residues joined by a peptide bond to form a polypeptide.
The terms "nucleic acid," "polynucleotide," and "oligonucleotide" refer to a polymer of nucleotides (e.g., ribonucleotides or deoxyribonucleotides), and include naturally occurring (adenosine, guanidine, cytosine, uracil, and thymidine), non-naturally occurring, and modified nucleic acids. The term is not limited by the length of the multimer (e.g., number of monomers). The nucleic acid may be single-stranded or double-stranded, and will typically contain 5'-3' phosphodiester linkages, although in some cases, the nucleotide analogs may have other linkages. Monomers are generally referred to as nucleotides. The term "non-natural nucleotide" or "modified nucleotide" refers to a nucleotide that contains a modified nitrogen-containing base, sugar, or phosphate group, or that incorporates a non-natural moiety in its structure. Examples of non-natural nucleotides include dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated and fluorophore-labeled nucleotides. The term "kit" refers to any article of manufacture (e.g., package or container) comprising at least one device that includes a solid support as described herein for specifically amplifying, capturing, labeling/converting or detecting a target nucleic acid sequence as described herein. The kit may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
The term "detect" and similar terms are used in this application to refer generally to a process or to discover or determine the presence or absence of something, as well as the degree, quantity, or level, or probability of occurrence. It is to be understood that the expressions "detecting the presence or absence", "detecting the presence or absence" and related expressions include both qualitative and quantitative detection. For example, quantitative detection includes determining the level, amount or quantity of SARS-CoV-2 associated nucleic acid sequence in the sample.
The invention relates to a polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 1.
The polypeptide comprises the following parts: S-Linker-N-avi-tag.
The S protein and the N protein are connected in series through the optimized rigid linker sequence, so that the two proteins have relatively independent space conformations, a plurality of dominant epitopes are added, and the sensitivity and the signal value are improved to a great extent; in addition, the fusion protein is introduced into the Avi-tag, so that the recombinant protein can be immobilized through the immobilized site, and the influence of steric hindrance brought by the coating process is reduced. Therefore, the polypeptide can achieve high sensitivity and specificity, and is not easy to miss detection. Through verification, six repeated rigid markers can better reach the design expectation, and the relative independence of the protein on the space structure is maintained. The Avi-tag protein is smaller, and enough unfolding space can be realized by the design of the C end, so that the coating efficiency of a solid phase such as magnetic beads is improved, and the application of the Avi-tag protein at a chemiluminescent end is better.
In some embodiments, the N-terminus of the polypeptide is further linked to a signal peptide.
In some embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 2.
In some embodiments, the polypeptide further comprises a tag protein attached to the C-terminus.
In some embodiments, the tag protein is a small molecular weight tag protein, e.g., having a molecular weight below 1kD or a number of amino acids of less than 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10).
In some embodiments, the Tag protein is selected from His, HA, c-Myc, aviTag, SNAP-Tag or Flag Tag.
In some embodiments, the His tag is 10 xhis, 9 xhis, 8 xhis, 7 xhis, or 6 xhis.
According to a further aspect of the invention, the invention also relates to a nucleic acid encoding a polypeptide as described above.
The nucleic acid may be DNA or RNA.
The invention also relates to vectors containing the nucleic acids as described above.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of 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 such that 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 Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).
In a specific embodiment, the vector is pCMV3.
According to a further aspect of the invention, the invention also relates to a host cell comprising a nucleic acid as described above, or a vector as described above.
In some embodiments, the host cell does not comprise a plant cell capable of developing into a whole plant, nor does the host cell comprise an animal cell capable of developing into a whole animal (e.g., germ cells, totipotent stem cells such as germ stem cells and embryonic stem cells, fertilized eggs).
In some embodiments, the host cell is a prokaryotic cell, such as e.coli.
In some embodiments, the host cell is a eukaryotic cell, such as a fungus (yeast, etc.).
In some embodiments, the host cell is an animal cell.
In some embodiments, the host cell is a mammalian cell.
In some embodiments, the host cell is a rodent cell, e.g., rat, mouse, hamster.
In some embodiments, the host cell is a primate cell, preferably a human.
In some embodiments, the host cell is a primary cell, such as a tumor cell, a liver cell, a heart muscle cell, a neuron, an endothelial cell, a stem cell, or the like.
In some embodiments, the host cell is a cell line;
common cell lines are for example:
cell lines derived from humans:
293、IMR-90、W1-38、A549、A431、BHL-100、BeWo、Caco-2、Chang、HCT-15、HeLa、HEK293(HEK293F)、HEp-G2、HEp-2、HT-1080、HT-29、JEG-2、MCF7、KB、Saos-2、WI-38、WISH、WS1、HUVEC、EB-3、Raji、IM-9、Daudi、H9、HL-60、Jurkat、K-562、U937、KG-1；
cell lines derived from mice:
McCoy、BALB/3T3、3T6、A9、AtT-20、Clone M-3、I-10、Y-1、WEHI-3b、ES-D3、F9；
hamster derived cell lines:
BHK-21、HaK、CHO-K1；
cell lines derived from rats:
AR42J、BRL3A、Clone 9、H4--Ⅱ-E-C3、GH1、GH3、IEC-6、L2、XC、LLC-WRC 256、Jensen、Rat2(TK-)、PC12、L6；
cell lines derived from other animals:
D-17、BT、MARC-145、CV-1、COS-1、COS-3、COS-7、Vero、B95-8、CRFK。
the invention also relates to a novel coronatine detection kit comprising a polypeptide as described above, an anti-antibody and a solid support;
the anti-antibody is an antibody against the species immunoglobulin from which the sample is derived;
the AviTag tag in the polypeptide is pre-biotinylated and conjugated to the solid support via the biotinylated AviTag.
In some embodiments, the solid support is a test tube, EP tube, multiwell plate, microplate well, bead or wafer.
In the present invention, the term "microparticle" may be a sphere, a spheroid, a cube, a polyhedron, or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 500 μm.
The fine particles are preferably magnetic fine particles, and the component thereof contains a magnetic substance. The magnetic substance can be metal (metal simple substance or alloy), nonmetal, or a compound formed by metal and nonmetal. Metals such as iron, alnico metals, and the like; nonmetallic materials such as ferrite nonmetallic materials (preferably Fe 2 O 3 Or Fe (Fe) 3 O 4 Magnetic nanoparticles); composites of metals and non-metals such as neodymium iron boron rubber magnetic composites.
The multiwell plate is preferably an ELISA plate which may contain 8, 16, 32, 48, 64, 96 or more well sites.
In some embodiments, the anti-antibody is conjugated to a signal substance.
In some embodiments, the detection kit for the new coronatine pneumonia may further include a sample pretreatment reagent (such as a sample purification enrichment reagent, a lysate, etc.), a washing solution (such as PBS, etc.), a buffer solution, a chromogenic reagent for a signal substance (such as horseradish peroxidase as a signal substance, ECL as a chromogenic reagent), etc.
The invention also relates to a new coronarium pneumonia detection test strip, which comprises a sample pad, a binding pad, a reaction membrane and an absorption pad, wherein a detection area and a quality detection area are arranged on the reaction membrane;
the conjugate pad is coated with an anti-antibody, and the anti-antibody is conjugated to a signal substance; the anti-antibody is an antibody against the species immunoglobulin from which the sample is derived;
the detection zone is conjugated with a polypeptide as described above; the AviTag tag in the polypeptide is pre-biotinylated and conjugated to the detection zone via the biotinylated AviTag.
In some embodiments, the immunoglobulin is at least any one of IgG, igM, or IgA.
In some embodiments, the anti-antibody is of a different species source than the species source of the sample being tested, and the following species sources may be selected as is common: rats, mice, dogs, goats, sheep, horses, donkeys, rabbits, chickens.
In some embodiments, the signal species is a metal particle, a fluorescent label, a chromophore label, an electron dense label, a chemiluminescent label, a radiolabel, or an enzyme.
In some embodiments, the signal substance is colloidal gold, fluorescein, fluorescent microspheres, acridinium esters, horseradish peroxidase, alkaline phosphatase, or β -galactosidase. These labels are listed in the following non-limiting section:
enzymes that produce detectable signals, such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds and dyes.
Groups having electron densities that can be detected by electron microscopy or by their electrical properties, such as conductivity, amperometry, voltage measurement, and resistance.
A detectable group, such as a molecule of sufficient size to induce a modification detectable in its physical and/or chemical properties; such detection may be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic force spectroscopy and tunneling).
Electronically dense substances, e.g. radioactive molecules (e.g 32 P， 35 S or 125 I)。
According to a further aspect of the invention, it also relates to the use of a polypeptide as described above for the preparation of a SARS-CoV-2 antibody detection test strip or kit.
In some embodiments, in the test strip or kit, the polypeptide is conjugated to the test strip, or to a solid support in the kit, via an AviTag tag protein.
In some embodiments, the test sample is selected from biological tissue or lavage fluid thereof, cells, body fluids, further selected from blood, serum, plasma, anticoagulation, cell culture supernatant, saliva, semen, amniotic fluid, villus, tissue or tissue lysate, pharyngeal swab, nasal swab, conjunctival swab, fecal specimen, stool, urine, bronchial lavage fluid, alveolar lavage fluid, sputum.
The subject for the above use may refer to a patient or an animal suspected of carrying SARS-CoV-2, in particular a mammal, e.g. bat, castors; preferably a primate, more preferably a human.
The recombinant antigen of the invention is used for preparing a novel coronavirus antibody detection kit or test strip, and the sensitivity and the detection rate of the reagent can be effectively improved.
The invention also relates to a method for detecting SARS-CoV-2 antibody, comprising:
SARS-CoV-2 antibody is detected using a polypeptide, kit or test strip as described above.
Embodiments of the present invention will be described in detail below with reference to examples.
EXAMPLE 1 expression cloning construction of SARS-CoV-2-S+N-pCMV3L
According to the figure 1, the amino acid sequence is selected to construct novel coronavirus S+N expression clone, and the linker sequence is EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK, namely rigid linker, which can obviously separate two proteins at a certain distance and maintain the relative independence of the proteins in space structure. The Avi-tag sequence is GLNDIFEAQKIEWHE, can carry out directional biotinylation modification, and is favorable for later development of high-sensitivity diagnostic reagents. The secretion signal peptide is a novel coronavirus S amino acid (Uniprot: P0DTC 2) sequence aa1-12, S is a novel coronavirus spike protein (Uniprot: P0DTC 2) amino acid sequence aa13-1273, and N is a novel coronavirus spike protein (Uniprot: A0A6C0T6Z 7) amino acid sequence aa1-419. The specific amino acid sequences for constructing the expression clones are shown below:
AT—MFVFLVLLPLVS—(sqcvnlttrtqlppaytnsftrgvyypdkvfrssvlhstqdlflpffsnvtwfhaihvsgtngtkrfdnpvlpfndgvyfasteksniirgwifgttldsktqsllivnnatnvvikvcefqfcndpflgvyyhknnkswmesefrvyssannctfeyvsqpflmdlegkqgnfknlrefvfknidgyfkiyskhtpinlvrdlpqgfsaleplvdlpiginitrfqtllalhrsyltpgdsssgwtagaaayyvgylqprtfllkynengtitdavdcaldplsetkctlksftvekgiyqtsnfrvqptesivrfpnitnlcpfgevfnatrfasvyawnrkrisncvadysvlynsasfstfkcygvsptklndlcftnvyadsfvirgdevrqiapgqtgkiadynyklpddftgcviawnsnnldskvggnynylyrlfrksnlkpferdisteiyqagstpcngvegfncyfplqsygfqptngvgyqpyrvvvlsfellhapatvcgpkkstnlvknkcvnfnfngltgtgvltesnkkflpfqqfgrdiadttdavrdpqtleilditpcsfggvsvitpgtntsnqvavlyqdvnctevpvaihadqltptwrvystgsnvfqtragcligaehvnnsyecdipigagicasyqtqtnsprrarsvasqsiiaytmslgaensvaysnnsiaiptnftisvtteilpvsmtktsvdctmyicgdstecsnlllqygsfctqlnraltgiaveqdkntqevfaqvkqiyktppikdfggfnfsqilpdpskpskrsfiedllfnkvtladagfikqygdclgdiaardlicaqkfngltvlpplltdemiaqytsallagtitsgwtfgagaalqipfamqmayrfngigvtqnvlyenqklianqfnsaigkiqdslsstasalgklqdvvnqnaqalntlvkqlssnfgaissvlndilsrldkveaevqidrlitgrlqslqtyvtqqliraaeirasanlaatkmsecvlgqskrvdfcgkgyhlmsfpqsaphgvvflhvtyvpaqeknfttapaichdgkahfpregvfvsngthwfvtqrnfyepqiittdntfvsgncdvvigivnntvydplqpeldsfkeeldkyfknhtspdvdlgdisginasvvniqkeidrlnevaknlneslidlqelgkyeqyikwpwyiwlgfiagliaivmvtimlccmtsccsclkgccscgscckfdeddsepvlkgvklhyt—EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK—msdngpqnqrnapritfggpsdstgsnqngersgarskqrrpqglpnntaswftaltqhgkedlkfprgqgvpintnsspddqigyyrratrrirggdgkmkdlsprwyfyylgtgpeaglpygankdgiiwvategalntpkdhigtrnpannaaivlqlpqgttlpkgfyaegsrggsqassrsssrsrnssrnstpgssrgtsparmagnggdaalalllldrlnqleskmsgkgqqqqgqtvtkksaaeaskkprqkrtatkaynvtqafgrrgpeqtqgnfgdqelirqgtdykhwpqiaqfapsasaffgmsrigmevtpsgtwltytgaiklddkdpnfkdqvillnkhidayktfpptepkkdkkkkadetqalpqrqkkqqtvtllpaadlddfskqlqqsmssadstqa—GLNDIFEAQKIEWHE)—HHHHHHHHHHH
SEQ ID NO. 1 is shown in brackets. After the amino acid is chemically synthesized, the amino acid is cloned into an expression vector pcMV3L to obtain an expression clone, and the vector diagram is shown in fig. 2.
EXAMPLE 2 recombinant protein expression and purification
The recombinant plasmid pCMV3L-SARS-CoV-2-S+N is transformed into E.coli Top10 competent cells, spread on LB plate containing 50ug/ml antibiotic Amp+ for culture, cultured overnight at 37 degrees, and single colony is selected and cultured in 1L LB medium containing 50ug/ml antibiotic Amp+. After 16-20h of culture at 37 ℃ and 200rpm, the thalli are collected by centrifugation, and a large amount of recombinant plasmids are prepared by using an endotoxin-free plasmid extraction kit, wherein the plasmid requires A 260 /A 280 ＝1.8-2.0。
The cells are recovered and cultured, the health of the cells is ensured before transfection, and the survival rate is higher than 90 percent. The cell density was transferred to 1.5-2.5X10 6 And each ml. Placing in a carbon dioxide culture shaking table for culture, wherein the culture shaking table is provided with: the temperature is 36.9 ℃, the carbon dioxide concentration is 5%, the rotating speed is 120rpm, and the culture is carried out for 2-4 hours. The amount of cells to be transfected is calculated, a certain amount of plasmid DNA and a corresponding amount of transfection reagent PEI are taken and diluted to a certain proportion by sterile PBS buffer solution respectively. Then adding diluted transfection reagent into diluted plasmid DNA, mixing quickly and evenly, standing for 10-20 min, adding dropwise into prepared cells, and carrying out suspension culture at 37 ℃ and 120 rpm. After culturing for 32-64h, adding feed liquid, continuing culturing for 32-64h, stopping culturing, centrifuging, and collecting supernatant, which is SARS-CoV-2-S+N antigen expression product.
The SARS-CoV-2-S+N recombinant antigen expression supernatant was filtered through a 0.45 μm filter membrane, and concentrated 5-10 times by using an ultrafiltration membrane bag having a pore size of 10kda to obtain a concentrated supernatant, which was purified. Loading into Ni affinity chromatographic column with pre-equilibrium, balancing 50 column volumes with balancing buffer (pH 8.0,100mM Tris-Cl,300mM NaCl), sequentially eluting with 20 times of balancing solution containing 20mM imidazole, linearly eluting with 20 times of column volumes (balancing solution containing 20-500mM imidazole), collecting eluate, detecting by electrophoresis (figure 3), collecting eluate containing target protein, dialyzing with dialysis bag with 10kda aperture for 12 hr, and dialyzing with exchange solution to obtain SARS-CoV-2-S+N sample.
EXAMPLE 3 Activity assay
The prepared SARS-CoV-2-S+N is used for sensitivity and specificity detection by using an Elisa method, antigen (SARS-CoV-2-S+N; SARS-CoV-2-S; SARS-CoV-2-N) is respectively coated, a new coronavirus diagnostic reagent chimeric antibody (self-production) is used for sensitivity detection, negative serum is used for specificity detection, then a mouse anti-human IgG-HRP is used for secondary antibody, a substrate is added for color development, and after reaction for 10min, a stop solution is used for stopping the reaction. The microplate reader reads the absorbance at wavelength 450:
1. diluting target antigen and control antigen with CB diluent to 5ug/ml and 10ug/ml, and taking 100ul of coated plates overnight (4 ℃);
2. cleaning the ELISA plate, adding sealing liquid (in lake for English-wound dilution) 150ul, and sealing at room temperature for about 5-24 h;
3. after washing, the ELISA plate is added with chimeric antibody (primary antibody), dilution gradient 10 3 ,10 4 ,10 5 Doubling, adding 100ul, and incubating at 37 ℃ for 1h;
serum was diluted 100-fold and incubated at 37℃for 1h with 100 ul;
4. after the ELISA plate is cleaned, adding mouse anti-human IgG (marked HRP), diluting by 1 ten thousand times, adding 100ul, incubating at 37 ℃ for 0.5h, and washing the plate;
5. adding 100ul of primer (TMD) after cleaning, and reacting for 10min;
6. adding a stop solution, and performing reading (double-numerical values 630 and 450) by using an enzyme-labeled instrument to preliminarily judge the activity condition.
Sensitivity analysis
Figure BDA0003232153760000121
Comparing Elisa data, the test signal value of SARS-CoV-2-S+N with the same coating amount of fusion protein is obviously higher than that of monomeric proteins SARS-CoV-2-N and SARS-CoV-2-S, and the sensitivity is greatly improved.
Specificity analysis
N S S+N
T1 0.441 0.236 0.214
T2 0.396 0.153 0.135
T3 1.116 0.441 0.163
T4 0.358 0.315 0.14
T5 0.295 0.272 0.171
T6 0.374 0.342 0.181
T7 0.887 0.446 0.167
T8 0.282 0.231 0.14
T9 0.333 0.285 0.299
T10 0.207 0.198 0.178
T11 0.709 0.335 0.213
T12 0.885 0.329 0.234
T13 0.446 0.213 0.104
T14 0.362 0.35 0.162
T15 0.853 0.656 0.184
T16 0.458 0.45 0.175
T17 0.308 0.27 0.177
T18 0.363 0.177 0.121
T19 0.982 0.151 0.106
T20 0.427 0.194 0.147
T21 0.311 0.228 0.18
T22 0.472 0.167 0.119
T23 0.374 0.15 0.123
T24 0.666 0.305 0.241
24 cases of negative physical blood tests show that a plurality of cases of false positive signals appear in SARS-CoV-2-S+N and the overall test value background is higher; and compared with the signal of negative physical examination blood, SARS-CoV-2-S+N has better specificity than SARS-CoV-2-S.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Shenzhen City Yahui Biotechnology Co., ltd
<120> polypeptide and its use in novel coronavirus detection
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<170> SIPOSequenceListing 1.0
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Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
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Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val
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Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
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Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
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Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
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Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
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Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
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Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
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Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
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Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
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<210> 2
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<212> PRT
<213> artificial sequence
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1 5 10

Claims (17)

1. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1;
the polypeptide comprises the following parts: S-Linker-N-avi-tag;
wherein the linker sequence is EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK.
2. The polypeptide of claim 1, wherein the N-terminus of the polypeptide is further linked to a signal peptide.
3. The polypeptide according to claim 2, wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO. 2.
4. The polypeptide of claim 1, wherein the C-terminal of the polypeptide is further linked to a tag protein.
5. The polypeptide of claim 4, wherein the tag protein is a His, flag, HA, c-Myc, aviTag, SNAP or Flag tag.
6. A nucleic acid encoding the polypeptide according to any one of claims 1 to 5.
7. A vector comprising the nucleic acid of claim 6.
8. A host cell comprising the nucleic acid of claim 6 or transformed with the vector of claim 7.
9. A kit for detecting a novel coronatine pneumonia, comprising the polypeptide, the anti-antibody, and the solid support according to any one of claims 1 to 5;
the anti-antibody is an antibody against the species immunoglobulin from which the sample is derived;
the AviTag tag in the polypeptide is pre-biotinylated and conjugated to the solid support via the biotinylated AviTag.
10. The detection kit for new coronatine pneumonia according to claim 9,
the solid support is a test tube, an EP tube, a multi-well plate, a well of a microplate, a bead or a wafer.
11. The detection kit for new coronatine pneumonia according to claim 9,
the anti-antibody is conjugated to a signal substance.
12. The new coronatine pneumonia detection test strip comprises a sample pad, a combination pad, a reaction membrane and an absorption pad, wherein a detection area and a quality detection area are arranged on the reaction membrane;
the conjugate pad is coated with an anti-antibody, and the anti-antibody is conjugated to a signal substance; the anti-antibody is an antibody against the species immunoglobulin from which the sample is derived;
the detection region is conjugated with the polypeptide of any one of claims 1-5; the AviTag tag in the polypeptide is pre-biotinylated and conjugated to the detection zone via the biotinylated AviTag.
13. The kit of claim 9, or the test strip of claim 12, wherein the immunoglobulin is at least any one of IgG, igM, or IgA.
14. The kit of claim 9, or the test strip of claim 12, the signal substance being a metal particle, a fluorescent label, a chromophore label, an electron-dense label, a chemiluminescent label, a radiolabel, or an enzyme.
15. Use of the polypeptide of any one of claims 1-5 in the preparation of a SARS-CoV-2 antibody detection test strip or kit.
16. The use according to claim 15, wherein the test sample is selected from biological tissue or lavage fluid, cells, body fluids thereof.
17. The use according to claim 16, wherein the test sample is selected from the group consisting of blood, serum, plasma, anticoagulation, cell culture supernatant, saliva, semen, amniotic fluid, villus, tissue or tissue lysate, pharyngeal swab, nasal swab, conjunctival swab, fecal specimen, fecal matter, urine, broncholavage, alveolar lavage, sputum.
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