CN115850399A - Thermostable spike protein and novel coronavirus antibody detection test strip - Google Patents
Thermostable spike protein and novel coronavirus antibody detection test strip Download PDFInfo
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Abstract
The invention provides a thermostable spike protein and a novel coronavirus antibody detection test strip, and belongs to the technical field of antibody detection. According to the invention, the structure of the spike protein is precisely modified, so that the problems that the detection result of the new crown antibody detection kit in the prior art is not accurate enough and has low correlation with a neutralizing antibody are solved. The thermostable spike protein provided by the invention successfully realizes the remarkable improvement of the stability and the yield of spike protein tripolymer, and the thermostable spike protein is used as a detection probe, so that the level of the new crown antibody in the peripheral blood of a human body can be rapidly and accurately tested, and the positive correlation of a positive antibody and a neutralizing antibody is better. The novel coronavirus antibody detection test strip provided by the invention is used as an in-vitro rapid diagnosis device, can be used for measuring the level of a new coronavirus antibody of a human body within 15 minutes by collecting a small amount of blood plasma or blood serum, is convenient and rapid to use, and has higher detection accuracy.
Description
Technical Field
The invention relates to the technical field of antibody detection, in particular to a thermostable spike protein and a novel coronavirus antibody detection test strip.
Background
The spike protein (spike protein) of the new coronavirus is composed of homotrimers, which play a key role in the invasion process of human body, and the spike protein is unstable, and particularly can generate a series of conformational changes when being combined with a receptor ACE2, and the change from a pre-fusion state (pre-fusion) to a post-fusion state (post-fusion) is the basis for the spike protein conformational change to play a biological function. Whether by vaccination or viral infection, spike proteins can cause a strong immune response in humans, inducing the human body to produce antibodies targeting spike proteins, some of which bind to spike proteins and block viral entry into the human body, known as neutralizing antibodies. Antibody levels (particularly levels of neutralizing antibodies) directly reflect the ability of the humoral immune part of the body to protect against viruses, and therefore, many researchers have conducted studies on antibody level tests for targeted spike proteins. The following can be reflected by testing the level of antibody targeting the spike protein: 1. whether a human is infected with a virus; 2. whether vaccination induces the human body to produce antibodies; 3. the antibody level of the human body is high or low.
Since the spike protein trimer is unstable and is easily damaged during purification, the yield is low when it is expressed by mammalian cells. At present, most of new crown antibody detection kits in the market only adopt a part (RBD or S1) of spike protein as a detection probe, and have the problem of underestimation of antibody level. Even if there are few detection kits using the intact spike protein as a probe, there is an underestimation phenomenon of antibody level in practical detection applications due to instability of the spike protein, and the correlation with neutralizing antibodies is low.
Disclosure of Invention
The invention aims to provide a thermostable spike protein and a novel coronavirus antibody detection test strip, and aims to solve the problems that a detection result of a novel coronavirus antibody detection kit in the prior art is not accurate enough and has low correlation with a neutralizing antibody.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a thermostable spike protein, the amino acid sequence of which is shown in SEQ ID NO. 1.
The invention also provides a gene of the heat-stable spike protein, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2.
The invention also provides a heat-stable spike protein recombinant expression vector, which comprises an initial vector and the gene for coding the heat-stable spike protein.
Preferably, the initial vector is pcDNA3.1 vector, and the gene encoding the thermostable spike protein is cloned between BamHI and XhoI cleavage sites of the recombinant vector.
The invention also provides a recombinant cell for expressing the heat-stable spike protein, and the recombinant cell is transfected with the heat-stable spike protein recombinant expression vector.
Preferably, the recombinant cell is a 293F cell or a CHO cell as an initial cell.
The invention also provides application of the heat-stable spike protein in preparation of a novel coronavirus antibody detection reagent.
The invention also provides a novel coronavirus antibody detection test strip which comprises a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad; the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and arranged on the bottom plate; the nitrocellulose membrane is sequentially provided with a detection line and a quality control line;
the colloidal gold pad is coated with a spike protein-colloidal gold compound, and the spike protein is the heat-stable spike protein;
the heat-stable spike protein is coated on the detection line;
the quality control line is coated with a goat anti-mouse antibody.
Preferably, on the nitrocellulose membrane, the detection line is close to the nearer side of the sample pad, and the quality control line is far away from the sample pad.
Preferably, the test strip still includes the card shell, the card shell includes back card and upper cover, the upper cover is equipped with fretwork test window and application of sample hole, detection line and quality control line are located under the test window, the sample pad is located under the application of sample hole.
The invention has the technical effects and advantages that:
the invention aims at the structure of the spike protein, carries out precise modification on the spike protein, successfully realizes the remarkable improvement of the stability and the yield of the spike protein tripolymer, takes the thermal stable spike protein as a detection probe, can quickly and accurately test the level of the new crown antibody in the peripheral blood of a human body, and has better positive correlation between a positive antibody and a neutralizing antibody. The novel coronavirus antibody detection test strip provided by the invention is used as an in-vitro rapid diagnosis device, can be used for measuring the level of a new coronavirus antibody of a human body within 15 minutes by collecting a small amount of blood plasma or blood serum, is convenient and rapid to use, and has higher detection accuracy.
Drawings
FIG. 1 shows the result of testing the thermal stability of spike protein;
FIG. 2 shows the results of a partial antibody level test;
FIG. 3 is a comparison of positive detection rates using only RBD and TS-spike protein;
FIG. 4 shows the correlation analysis results of the positive antibody and the neutralizing antibody.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Sources of experimental samples:
sample type: human plasma (with plasma that has never been infected with new corona virus and has not been vaccinated with new corona vaccine as a negative control) originated from the seventh hospital affiliated with Zhongshan university (Shenzhen). Plasma samples were collected in containers containing EDTA anticoagulant.
Collecting samples: the process of collecting and detecting the blood sample of the patient needs to be operated according to the latest edition of the laboratory detection technical guideline for pneumonia infected by novel coronavirus issued by the State health Commission.
Sample preservation: separating the sample in time for detection after blood sample collection; if the detection cannot be carried out in time, the sample can be stably stored for 8 days under the storage condition of 2-8 ℃, can be stored for a long time below 18 ℃ below zero, and is repeatedly frozen and thawed for no more than 5 times. The detection can be carried out using a sample inactivated at 56 ℃ for 30 minutes.
Sample safety: all samples were considered potentially infectious items and were performed strictly in accordance with national relevant standards and guidelines.
Example 1 spike protein engineering and expression vector construction
The spike protein structure is precisely reconstructed based on the structure of the spike protein (published spike protein structure (PDB: 6 VXX)), and the specific method is as follows:
performing G413C and V987C mutation respectively to form a pair of disulfide bonds between S1 and S2 of the spike protein, simultaneously deleting Furin enzyme cleavage site (PRRA), removing original transmembrane region, and replacing with Foldon sequence; <xnotran> 817, 892, 899 942 P, MGILPSPGMPALLSLVSLLSVLLMGCVAETGTQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPCQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKCEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKGSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH, SEQ ID NO.1 , GCCACCATGGGGATTCTGCCTAGCCCCGGCATGCCCGCCCTCCTGAGCCTGGTGAGCCTGCTGAGCGTCCTGTTGATGGGGTGCGTGGCCGAGACCGGCACACAGTGCGTGAACCTGACAACTAGAACACAGCTGCCCCCAGCCTACACCAACAGCTTTACTAGGGGAGTGTACTACCCTGATAAGGTGTTCAGGAGCAGTGTGCTGCACAGCACCCAGGACCTCTTTTTGCCCTTTTTCAGTAACGTGACCTGGTTCCACGCTATCCACGTGAGCGGCACAAACGGCACAAAACGGTTCGATAACCCAGTGCTGCCCTTCAACGACGGCGTCTACTTCGCCAGCACTGAGAAGTCCAACATTATCAGGGGGTGGATCTTCGGGACAACCCTGGACAGCAAGACACAGAGCCTCCTGATTGTGAACAACGCCACCAACGTGGTCATTAAGGTCTGCGAGTTCCAGTTCTGCAACGACCCCTTTCTGGGGGTGTACTACCACAAGAACAACAAGTCCTGGATGGAGTCCGAGTTTCGGGTGTACTCCAGCGCCAACAACTGCACATTTGAGTACGTGAGCCAGCCCTTCTTGATGGATCTGGAGGGCAAGCAGGGGAACTTTAAGAACCTTCGGGAGTTCGTCTTTAAGAACATCGACGGGTACTTTAAGATCTACAGCAAGCACACCCCTATTAACCTGGTCCGCGATCTGCCCCAGGGCTTCAGCGCCCTGGAGCCACTGGTGGACCTCCCCATCGGGATCAACATTACTAGGTTCCAGACACTCCTGGCCCTGCACCGGAGCTACCTGACTCCCGGCGATAGCTCCAGCGGGTGGACTGCCGGGGCCGCCGCCTACTACGTCGGCTACCTGCAGCCCAGGACATTCCTGCTCAAGTACAACGAGAACGGCACCATCACTGATGCCGTGGACTGCGCTCTCGATCCCCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAGAAGGGGATCTACCAGACATCTAACTTCAGGGTGCAGCCCACTGAGTCCATTGTGAGATTTCCTAACATCACCAACCTGTGCCCCTTCGGCGAGGTCTTTAACGCCACACGGTTCGCCAGCGTCTACGCTTGGAACAGGAAGCGGATCTCCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCTCCTTCAGCACCTTTAAGTGCTACGGCGTGAGCCCCACTAAGCTGAACGACCTGTGCTTCACTAACGTGTACGCTGATAGCTTCGTGATTAGGGGCGATGAGGTCCGGCAGATTGCCCCCTGCCAGACAGGGAAGATCGCTGACTACAACTATAAGCTGCCCGATGACTTCACAGGGTGTGTGATCGCCTGGAACAGCAACAACCTGGATAGCAAGGTCGGGGGGAACTACAACTACCTGTACAGGCTGTTCAGAAAGTCCAACCTGAAGCCCTTCGAGCGGGATATCAGCACTGAGATCTACCAGGCCGGGAGCACCCCCTGCAACGGCGTCGAGGGCTTTAACTGCTACTTTCCCCTCCAGAGCTACGGCTTCCAACCCACCAACGGCGTGGGGTACCAGCCCTACCGGGTCGTGGTGCTTAGCTTCGAGCTCCTGCACGCCCCTGCAACCGTGTGCGGCCCCAAGAAGAGCACAAACCTCGTGAAGAACAAGTGCGTGAACTTCAACTTCAACGGGCTGACCGGCACCGGCGTGCTGACTGAGAGTAACAAGAAGTTTCTCCCCTTCCAGCAGTTCGGGAGGGATATTGCCGACACTACTGATGCCGTGCGCGACCCTCAGACCCTTGAGATTCTGGACATCACTCCCTGCTCCTTCGGCGGAGTGTCCGTGATCACACCAGGGACCAACACTAGCAACCAGGTCGCCGTCTTGTACCAGGACGTGAACTGCACCGAGGTGCCCGTGGCCATCCACGCCGATCAGCTGACCCCAACTTGGCGGGTGTACTCCACCGGGAGCAACGTGTTTCAGACACGGGCCGGGTGCCTGATCGGGGCTGAGCATGTCAACAACAGCTACGAGTGCGATATTCCTATCGGCGCCGGGATTTGCGCCAGCTACCAGACACAGACAAACAGCAGGTCCGTCGCCAGCCAGAGCATCATCGCCTACACAATGTCCTTGGGGGCCGAGAACAGCGTCGCCTACAGCAACAACAGCATTGCCATCCCCACCAACTTCACAATCAGCGTCACAACCGAGATCCTGCCCGTGAGCATGACTAAGACATCCGTGGATTGCACAATGTACATCTGCGGCGACTCCACAGAGTGCAGTAACCTGCTGCTCCAGTACGGGTCCTTTTGTACCCAGCTGAACAGAGCCTTGACCGGCATTGCCGTGGAGCAGGACAAGAACACCCAGGAGGTGTTTGCCCAGGTGAAGCAGATCTACAAGACACCCCCCATAAAGGACTTCGGGGGCTTTAACTTCTCCCAGATCTTGCCCGACCCCAGCAAGCCCTCCAAGCGGTCACCTATCGAGGATCTGCTGTTCAACAAGGTCACCCTGGCCGATGCCGGCTTTATCAAACAGTATGGCGATTGCCTGGGCGACATAGCCGCCAGAGATCTGATCTGCGCCCAGAAATTCAACGGCCTGACAGTTCTCCCACCTCTGCTGACCGACGAGATGATCGCTCAGTACACCTCTGCCCTGCTGGCTGGCACCATCACATCTGGGTGGACATTTGGCGCCGGCCCCGCCCTGCAGATCCCCTTTCCCATGCAGATGGCCTATAGATTCAACGGAATCGGCGTGACCCAGAACGTGCTGTATGAAAACCAGAAGCTGATCGCTAACCAGTTCAATTCTGCCATCGGCAAGATCCAGGACTCCCTCTCCTCTACCCCCTCCGCCCTGGGGAAGTTGCAGGATGTGGTGAACCAGAACGCCCAGGCCCTGAACACACTGGTGAAGCAGCTGAGCAGCAACTTTGGCGCCATCAGTAGCGTCCTGAACGACATCCTGTCACGGCTTGATAAGTGCGAGGCCGAGGTCCAGATCGATCGGCTGATTACAGGCCGGCTGCAGAGTCTCCAGACCTACGTCACCCAGCAGCTGATCCGGGCTGCCGAGATTAGAGCCTCCGCCAATCTCGCCGCCACAAAGATGAGTGAATGCGTCCTGGGCCAGAGCAAAAGGGTCGACTTTTGCGGGAAGGGGTACCACCTGATGTCCTTTCCCCAGTCCGCCCCTCACGGAGTGGTGTTCCTCCATGTCACATACGTGCCTGCCCAGGAGAAGAACTTCACTACAGCCCCCGCCATTTGCCACGATGGGAAGGCCCATTTCCCTAGAGAGGGCGTGTTCGTGTCCAACGGGACCCACTGGTTCGTGACTCAGCGGAACTTTTATGAACCCCAGATTATCACAACCGATAACACATTTGTCTCCGGCAACTGTGATGTGGTCATCGGCATCGTGAACAACACCGTGTACGACCCACTGCAGCCTGAGCTTGACAGCTTTAAGGAGGAGCTGGACAAGTACTTTAAGAACCACACCAGCCCTGATGTGGACCTTGGCGACATCAGCGGCATTAACGCCAGCGTGGTGAACATTCAGAAGGAGATCGATAGACTGAACGAGGTGGCCAAGAACTTGAACGAGTCACTGATTGACCTGCAGGAGCTGGGGAAGTACGAGCAGTACATTAAGGGGTCCGGGAGGGAGAACCTGTACTTTCAGGGGGGGGGGGGGTCCGGCTACATTCCCGAGGCCCCAAGGGATGGCCAGGCCTACGTGAGGAAGGATGGGGAGTGGGTGCTGCTGAGCACCTTCTTGGGACACCACCACCACCACCATTAA </xnotran> As shown in SEQ ID NO. 2.
The gene expressed by the modified protein is subjected to codon optimization aiming at a human-derived expression system, and then is synthesized on a mammalian cell expression vector pcDNA3.1, particularly between BamHI and XhoI enzyme cutting sites of the vector, and the step is finished by Nanjing Kingsry Biotech Co.
Example 2 construction of supercoiled plasmid, transfection of cells
High quality supercoiled plasmid was extracted using MN endotoxin free plasmid macroextraction kit for transfection of 293F or CHO cells:
the target sequence was amplified by PCR, and after successful PCR amplification was identified by 1% agarose gel, the template containing methylated protoplasmids was digested by enzyme digestion with DpnI. 10 mu L of the PCR product after enzyme digestion of DpnI is taken to be transformed into 100 mu L of DH5 competence (purchased from Shenzhen kang Life science and technology Co., ltd.), ice bath is 30min, heat shock is carried out for 60s at 42 ℃, ice bath is carried out for 2min, 900 mu L of non-antibiotic LB is added, shaking table is carried out at 37 ℃ for 45min, the transformed competence is centrifuged at 4000rpm for 3min, 900 mu L of culture medium is removed by suction, and the rest 100 mu L of heavy suspension bacteria are leftThe resulting mixture was applied to 100. Mu.g/ml Ampicillin (Amp) + ) On the LB plate of (5), the plate was cultured overnight in an inverted incubator at 37 ℃. Single colonies were picked from the plate and added to 5ml of liquid LB containing 100ug/ml Amp +, shaken overnight (220 rpm) at 37 ℃ and the plasmid was extracted the next day for sequencing and identification, which was done by Bio-technology, inc., boxing, beijing Rui, guangzhou, inc.
Expi293F TM Cells (ThermoFisher, cat: A14527) were treated with Expi293 TM Expression Medium (ThermoFisher, cat: A1435101) was passaged and expressed in SMM 293-TII Expression Medium (Sino Biological, cat: M293 TII). Cells were diluted to 2-3X 10 the day before transfection 6 cells/ml, volume is calculated from the total volume to be expressed. Ensuring a cell density of 3X 10 at transfection 6 cells/ml。
The transfection medium was in Opti-MEM (ThermoFisher, cat: 31985070) and pre-dispensed at the required amount and pre-warmed to 37 ℃; PEI was used for transfection with PEI 40K (1 mg/ml, directly dissolved in 0.22um cold filtered water, pH adjusted to 7.0 with 1M NaOH) DNA: PEI =1:2.7.
the transfection procedure was as follows:
preparing a 50ml sterile centrifuge tube A, adding DNA into an Opti-MEM culture medium, and uniformly mixing the DNA with a final volume of 50 ml; preparing a 50ml sterile centrifuge tube B, adding PEI 40K into an Opti-MEM culture medium, and mixing uniformly, wherein the final volume is 50 ml; standing and incubating the two solutions at room temperature for 5min; preparing a 125ml flash, transferring the liquid in the centrifuge tube A into a cell shake flask, then adding the liquid in the centrifuge tube B into the cell shake flask dropwise, shaking while dropwise adding, standing and incubating at room temperature for 20min, adding the mixed solution of DNA-PEI dropwise into the cells to complete transfection, placing the transfected cells at 32 deg.C, and 5% CO 2 Culturing at 125rpm, and adding 1% supplement (SANYO. Zhang-SMS 293-SUPI medium) to each of Day1, day 3 and Day 5.
EXAMPLE 3 protein purification
When the cell growth activity rate is reduced to 85-90%, centrifuging to collect cell supernatant (4-9000 rpm-90 min). Binding buffer (25mM phosphate buffer, pH 8.0, 300mM NaCl, 10mM imidazole, 0.5mM PMSF) was added to a magnetic stirrer with stirring, and the cell supernatant was filtered with suction through a1 μm filter. At 4 deg.C, a peristaltic pump at 2.5ml/rpm was applied to the talon column and circulated overnight. 30CV were washed on AKTA with buffer A (25mM phosphate buffer, pH 8.0, 300mM NaCl, 10mM imidazole, total pH 8.01). 5ml/min,15 ml/tube collection; the elution was carried out on AKTA with a gradient of 0-100% buffer B (25mM phosphate buffer, pH 8.0, 300mM NaCl, 500mM imidazole, total pH 8.01) at 5ml/min,5 ml/tube collection. SDS-PAGE identifies concentration in 100kDa concentration tubes. And (3) performing Gel filtration purification on the concentrated protein (the column is superdex 200increatase 10/300), replacing buffer into PBS (pH 7.4), and concentrating to 1-2 mg/ml by using a new 100kDa concentration tube to obtain the modified heat-stable spike protein, and freezing and storing in a refrigerator at-80 ℃.
Example 4 preparation of test paper for detecting Neocrown antibody
The novel coronavirus (2019-nCoV) total antibody in serum is detected by adopting a colloidal gold double-antigen sandwich method measuring principle. Pre-coating a colloidal gold-labeled new crown recombinant protein S antigen (Au-Ag 1) on a glass cellulose membrane, respectively coating a detection line and a quality control line on a nitrocellulose membrane with a new crown recombinant protein S antigen (Ag 2) and an antibody, when a sample is positive, combining the new crown antibody in the sample with the colloidal gold-labeled new crown recombinant protein S antigen to form a complex, moving the complex forwards along a paper strip due to chromatography, forming an immune complex with the pre-coated new crown recombinant protein S antigen (Ag 2) for coagulation and color development when passing through the detection line, and coagulating and color development of a free gold marker on the quality control line; negative results only developed the quality control line.
The equipment used was: peak-and-flight film metal spraying instrument, model HGS510; a numerical control high-speed cutting machine, model ZQ-600; electric heating air blast drying box, DHG-9240A; digital display constant temperature magnetic stirrer, FK-H1; small centrifuges, LX-800; high speed refrigerated centrifuges were purchased from Thermo fisher.
The reagents used were: the thermostable spike protein (TS-spike protein) prepared in the above example, the New coronavirus S protein antigen (purchased from Meyer' S medical treatment) at a concentration of 1.1mg/ml, the goat anti-mouse antibody 0.5mg/ml, the murine IGG; calf serum albumin; chloroauric acid; trisodium citrate; BASE1 buffer; potassium carbonate solution (pH adjusted).
The materials used were: j270 absorbent paper; 8975/Z80B glass fiber; sartorius 95 nitrocellulose membrane; a 32pvc backplane; a plurality of packing materials such as aluminum foil packing bags, drying agents and the like.
The preparation method comprises the following steps:
preparing colloidal gold: taking a 1000mL clean conical flask, adding 500mL of 0.02% tetrachloroauric acid solution into the conical flask, opening a heating device, preheating, placing the conical flask on the heating device for heating until boiling, continuously stirring by magnetic force during the heating, quickly adding 1.5mL of 10% sodium citrate solution into the conical flask, continuously shaking up, boiling for 5min, closing the heating device, and naturally cooling the solution to room temperature for later use.
Labeling with colloidal gold: to 50ml of colloidal gold, 400. Mu.L of potassium carbonate solution was added to adjust the pH, magnetic stirring was maintained, 0.5mg of TS-Spike protein was added, stirring was carried out for 5 minutes, and 500. Mu.L of 10% BSA was added as a stabilizer.
Purifying the colloidal gold immune complex: firstly, centrifuging at low speed for 10 minutes at 5000 rpm; 8000 rpm for 20 minutes; the precipitate is combined, collected and redissolved to one twentieth of the original volume for later use.
Preparing a colloidal gold pad: labeled TS-Spike protein colloidal gold complex, diluted to OD by using Base1 540 =2.27, soaking the glass fiber Z80B, and drying the glass fiber at 37 ℃ for standby.
Scribing a film: coating antigen TS-Spike protein on a nitrocellulose membrane by using a scribing tool as a detection line (T line), coating goat anti-mouse antibody as a quality control line (C line), and drying at 37 ℃ for later use.
Sample pad: 8975 glass fibers were used and cut into 1.5cm wide strips for use.
Assembling: sequentially sticking J270 absorbent paper, a nitrocellulose membrane coated with TS-Spike protein and goat anti-mouse antibody, a colloidal gold combined pad coated with TS-Spike protein and an 8975 glass fiber sample pad onto a PVC (polyvinyl chloride) bottom plate, cutting into strips, putting into a card shell, transferring into an aluminum foil bag together with a drying agent, sealing, and storing at normal temperature for later use.
Detection method and determination result:
and (3) dropwise adding 20 mu L of the plasma sample into a colloidal gold detection hole, adding 40 mu LPBS, standing for 15min, and recording the result. When the C line of the quality control line has a strip, the test strip for detecting the neocorona antibody is effective. When the detection line T line has a strip, the sample contains the new coronavirus antibody, and if the detection line T line has no strip, the sample does not contain the new coronavirus antibody or the content is too low to be detected.
Experimental example 1 thermal stability test
To test the stability of spike proteins before and after modification, we adopted the following two methods: the method comprises the steps of placing fresh purified spike protein before modification and after modification at 4 ℃, sampling respectively at 0 day, 1 day, 3 days and 30 days to prepare negative staining samples, observing the structure and aggregation state of the protein through a negative staining electron microscope, and taking the negative staining samples as one of the evaluation standards of the protein stability. The second method is that the fresh purified spike protein before and after modification is placed at 60 ℃ for 30min, then negative staining sample preparation is carried out, the structure and aggregation state of the protein are observed through a negative staining electron microscope, and the structure and aggregation state are used for evaluating the thermal stability of the protein.
The thermal stability of the modified spike protein and the original spike protein before modification was tested, and the results are shown in fig. 1. Fig. 1 shows that the thermal stability of the modified spike protein is significantly improved, and the spike protein can still maintain the state of trimer after being placed at 60 ℃ for 30 minutes.
Experimental example 2 yield measurement
The yield detection is carried out on the modified spike protein and the original spike protein before modification, and the result is as follows:
and (3) measuring the ultraviolet absorption value of the protein by an ultraviolet absorption method, substituting the molar extinction coefficient of the Spike protein into the ultraviolet absorption value according to the Lambert beer law, namely A = epsilon c l), so as to obtain the concentration (mg/ml) of the protein, and multiplying the concentration by the volume of the protein to obtain the total mass of the protein. The result shows that the yield of the Spike protein before modification is only 1mg/L due to instability, and the yield of the TS-Spike after modification is improved to be not less than 5mg/L, thereby realizing remarkable improvement.
Experimental example 3 application of test strip for detecting neocoronatine
The test paper strip for detecting the new crown antibody prepared in the embodiment 4 is used for detecting the antibody, the sample source is as above, the detection result of part of the sample is shown in figure 2, and as can be seen from figure 2, the test paper strip for detecting the antibody assembled by the TS-Spike provided by the invention can clearly reflect the antibody level of a human body inoculated with the new crown vaccine and can present different results of negative → strong positive.
Experimental example 4 statistics of Positive detection Rate
Using test paper strips using RBD as probes as a control group, 31 samples (each inoculated with one or two doses of vaccine, and a part of individuals inoculated with the vaccine and sampled at a short time interval) were tested, and the positive detection rate was counted, with the results shown in fig. 3. The result shows that the kit using the RBD probe is only 16.13% positive, and the TS-Spike test strip provided by the invention has a positive detection rate of 38.71%.
Experimental example 5 correlation test between Positive antibody and neutralizing antibody
The results of 48 of the tests were normalized for neutralizing antibody level, anti-S antibody level and anti-ts S IgG antibody level and are shown in fig. 4, panel a. 148 cases of anti-S IgG antibody level and anti-tsS IgG antibody level were also counted separately, and the results are shown in FIG. 4B and FIG. 4C, respectively. The result shows that the correlation between the positive antibody and the neutralizing antibody is higher when the TS-Spike provided by the invention is used for detecting the new crown antibody.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A thermostable spike protein, characterized in that the amino acid sequence of said thermostable spike protein is shown in SEQ ID No. 1.
2. The gene encoding the thermostable spike protein of claim 1, wherein the nucleotide sequence of said gene is represented by SEQ ID No. 2.
3. A thermostable spike protein recombinant expression vector comprising an initial vector and the gene encoding a thermostable spike protein according to claim 2.
4. The recombinant expression vector of claim 3, wherein the initial vector is pcDNA3.1 vector, and the gene encoding the thermostable spike protein of claim 2 is cloned between BamHI and XhoI cleavage sites of the recombinant vector.
5. A recombinant cell expressing the thermostable spike protein of claim 1, wherein said recombinant cell is transfected with a thermostable spike protein recombinant expression vector of claim 3 or 4.
6. The recombinant cell of claim 5, wherein the recombinant cell is a 293F cell or a CHO cell as a starting cell.
7. Use of the thermostable spike protein of claim 1 in the preparation of novel coronavirus antibody detection reagents.
8. A novel coronavirus antibody detection test strip is characterized by comprising a bottom plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad; the sample pad, the colloidal gold pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and arranged on the bottom plate; the nitrocellulose membrane is sequentially provided with a detection line and a quality control line;
the colloidal gold pad is coated with a spike protein-colloidal gold complex, wherein the spike protein is the heat-stable spike protein of claim 1;
coating the detection line with the thermostable spike protein of claim 1;
and the quality control line is coated with a goat anti-mouse antibody.
9. The novel coronavirus antibody detection test strip of claim 8, wherein the detection line is close to the sample pad and the quality control line is far from the sample pad on the nitrocellulose membrane.
10. The novel coronavirus antibody detection test strip of claim 8 or 9, wherein the test strip further comprises a card shell, the card shell comprises a back card and an upper cover, the upper cover is provided with a hollow test window and a sample adding hole, the test line and the quality control line are positioned below the test window, and the sample pad is positioned below the sample adding hole.
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