CN111830258A - Novel coronavirus specific antibody detection kit - Google Patents
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- CN111830258A CN111830258A CN202010630413.3A CN202010630413A CN111830258A CN 111830258 A CN111830258 A CN 111830258A CN 202010630413 A CN202010630413 A CN 202010630413A CN 111830258 A CN111830258 A CN 111830258A
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention relates to a novel coronavirus specific antibody detection kit, which comprises a kit body and test paper, wherein the test paper is arranged in the kit body, the test paper comprises a PVC (polyvinyl chloride) plate, a sample pad, a colloidal gold pad, an NC (numerical control) membrane and absorbent filter paper, the sample pad, the colloidal gold pad, the NC membrane and the absorbent filter paper are positioned on the PVC plate and are sequentially butted from back to front, a detection line T and a quality control line C are arranged on the NC membrane, a sampling window corresponding to the sample pad and a display window corresponding to the NC membrane are arranged on the kit body, and a detection mark T and a quality control mark C which respectively correspond to the detection line T and the quality control line C. By adopting the technical scheme, the invention provides the novel coronavirus specific antibody detection kit, and the test paper comprises a PVC plate, a sample pad, a colloidal gold pad, an NC membrane and water absorption filter paper, wherein the sample pad, the colloidal gold pad, the NC membrane and the water absorption filter paper are positioned on the PVC plate and are sequentially butted from back to front, so that the test effect is more obvious, and the test is more accurate and quicker.
Description
Technical Field
The invention relates to the field of medical use, in particular to a novel coronavirus specific antibody detection kit.
Background
Coronaviruses are a large family of viruses belonging to the phylogenetic group of the family of Coronaviridae (Coronaviridae) and the genus Coronavirus (Coronavirus). The coronavirus is a positive strand single strand RNA virus with an outer mantle (envelope), the diameter of the coronavirus is about 80-120 nm, the genetic material of the coronavirus is the largest of all RNA viruses, and only human, mouse, pig, cat, dog and poultry vertebrates are infected. Coronavirus particles are irregular in shape, and the virions are coated with a fat membrane, and the surface of the membrane has three glycoproteins: spike glycoprotein (S, Spike Protein, which is the receptor binding site, cytolytic and major antigenic site); small Envelope glycoprotein (E, Envelope Protein, smaller, Envelope-bound Protein); membrane glycoproteins (M, Membrane proteins) are responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope). A few species also have hemagglutinin glycoproteins (HE proteins). The nucleic acid of coronavirus is non-segmented single-stranded (+) RNA, has the length of 27-31kb, is the longest RNA nucleic acid chain in RNA virus, and has important structural characteristics specific to positive-strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, and the 3' end is provided with a polyA tail structure. This structure is very similar to eukaryotic mRNA and is an important structural basis for the genomic RNA itself to function as a translation template, and the RNA-DNA-RNA transcription process is omitted. The recombination rate between RNA and RNA of coronavirus is very high, and the variation of virus is caused by the high recombination rate, and after recombination, the RNA sequence is changed, so that the amino acid sequence coded by nucleic acid is changed, and the protein formed by amino acid is changed, so that the antigenicity of the protein is changed. The result of the change of antigenicity is that the original vaccine is ineffective and the immunity fails.
The novel coronavirus pneumonia, namely '2019-nCoV', has the main manifestations of fever, hypodynamia and dry cough. A few patients have nasal obstruction, watery nasal discharge, diarrhea, etc. In severe cases, dyspnea occurs after one week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis and the appearance of blood coagulation dysfunction. Currently, there is no specific therapeutic agent for the new coronavirus pneumonia, and only a part of antiviral drugs are recommended to be tried for the treatment of the new coronavirus, such as lopinavir/ritonavir, alpha-interferon, etc., based on the early SARS and MERS drug research and small sample clinical treatment examples. Therefore, early diagnosis, cutting of viral transmission pathways and prevention of viral spread are extremely important.
24 days 1 month in 2020, the virus disease prevention and control center of China successfully separates the first strain of virus seed information in China and important authority information such as an electron microscope photo, a novel coronavirus nucleic acid detection primer and a probe sequence. And then, novel coronavirus strains are also successively separated from infectious disease national key laboratories affiliated to the Zhejiang province disease prevention and control center and the Zhejiang university medical college and the first hospital. Based on the sequence information, researchers at home and abroad can put forward nucleic acid molecular diagnosis products at the first time, basically select novel coronavirus conservative sequence design probes and primers, and realize the positive and negative differentiation of samples through a fluorescent PCR technology. The method has good detection sensitivity and high accuracy, is suitable for the definite diagnosis of the novel coronavirus, but has the following defects: 1. operators who need professional training and special operation sites are needed, otherwise, false positive is extremely high; 2. professional equipment, namely a fluorescent quantitative PCR instrument is needed, the equipment has a high value, and is difficult to bear by primary hospitals; 3. the detection time is longer, and at least 1.5 hours are needed.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects of the prior art, the invention provides a novel detection kit for specific antibodies of coronavirus, which can be used at home, has low cost and simple operation steps, can be used for detecting at home and has short detection time.
The technical scheme of the invention is as follows: a novel coronavirus specific antibody detection kit, comprising: including box body and test paper, the test paper is installed in the box body, the test paper is including the PVC board, be located the PVC board and by sample pad, colloidal gold pad, NC membrane and the filter paper that absorbs water of backward preceding butt joint in proper order, and the epimembranal detection line T and quality control line C that is equipped with of NC, be equipped with the sampling window that corresponds the sample pad on the box body and correspond the display window of NC membrane, on the box body and be equipped with respectively with detection line T and the detection sign T and the quality control sign C that quality control line C corresponds at the display window side.
Preferably, the front end of the sample pad presses the back end of the colloidal gold pad on the PVC board, the front end of the colloidal gold pad presses the NC membrane on the PVC board, and the back end of the water-absorbing filter paper presses the front end of the NC membrane on the PVC board.
Preferably, the box body form first inferior valve and the upper cover that holds the chamber and the chamber is held to the second including interconnect, be equipped with first wall body on the inferior valve, cover on and be equipped with the second wall body, first wall body and second wall body butt joint hold the chamber with first chamber and the second and hold the chamber and separate, the test paper install and first hold the intracavity, sampling window, display window, detection sign T and matter accuse sign C set up on covering, hold the intracavity at the second and have installed the sampling needle, be formed with between upper cover rear end and inferior valve rear end and supply the sampling needle to get the mouth of getting of putting get put get the mouth department and install the installation piece, sampling needle afterbody fixed with the installation piece.
Preferably, the sampling window is inwards recessed to form a conical sampling cavity, and a hole for blood sample in the cavity to be dripped into a sampling area of the test paper is formed in the bottom wall of the sampling cavity.
Preferably, the sampling window and the display window are covered with films.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the test paper comprises a PVC plate, a sample pad, a colloidal gold pad, an NC membrane and water absorption filter paper, wherein the sample pad, the colloidal gold pad, the NC membrane and the water absorption filter paper are positioned on the PVC plate and are sequentially butted from back to front, so that the test effect is more obvious, and the test is more accurate and faster; the conical sampling cavity can effectively stabilize the blood sample of a tester during detection, so that the blood sample is not easy to diffuse outwards; the second holds the chamber, and the intracavity is held at the second to the sampling needle, simple structure, and the sampling needle of being convenient for is taken along with the usefulness.
Drawings
FIG. 1 is a block diagram of an embodiment of the present invention;
FIG. 2 is an exploded view of the cartridge in an embodiment of the invention;
FIG. 3 is a diagram illustrating the structure of the test strip according to one embodiment of the present invention;
FIG. 4 is a diagram of the analysis of the major epitopes of the S antigen;
FIG. 5 is a graph of E antigen major epitope analysis;
FIGS. 6 and 7 are graphs of the major epitopes of the M antigen.
The test paper box comprises a box body 100, test paper 200, a PVC plate 210, a sample pad 220, a colloidal gold pad 230, an NC film 240, absorbent filter paper 250, a detection line T241, a quality control line C242, a sampling window 110, a display window 120, a lower shell 130, an upper cover 140, a first wall body 131, a second wall body 141, a sampling needle 300, a pick-and-place port 310, a mounting block 320, a sampling cavity 111 and a hole 112.
Detailed Description
The technical solutions in the embodiments will be described clearly and completely with reference to the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all the directional indications (such as up, down, front, and back … …) in the description of the present invention are only used to explain the relative position relationship between the components, the movement, etc. in a specific posture (as shown in the drawings), and if the specific posture is changed, the directional indication is changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated.
As shown in fig. 1-3, the novel coronavirus specific antibody detection kit of the present invention comprises a kit body 100 and a test paper 200, wherein the test paper 200 is installed in the kit body 100, the test paper 200 comprises a PVC plate 210, a sample pad 220, a colloidal gold pad 230, an NC membrane 240 and a water-absorbing filter paper 250, the sample pad 220 is located on the PVC plate 210 and is sequentially butted from back to front, a detection line T241 and a quality control line C242 are arranged on the NC membrane 240, a sampling window 110 corresponding to the sample pad 220 and a display window 120 corresponding to the NC membrane 240 are arranged on the kit body 100, and a detection mark T and a quality control mark C corresponding to the detection line T241 and the quality control line C242 are arranged on the side of the display window 120 on the kit body 100.
Preferably, the front end of the sample pad 220 presses the back end of the colloidal gold pad 230 against the PVC plate 210, the front end of the colloidal gold pad 230 presses the NC film 240 against the PVC plate 210, and the back end of the absorbent filter paper 250 presses the front end of the NC film 240 against the PVC plate 210.
Preferably, the box body 100 includes a lower shell 130 and an upper cover 140 which are connected to each other to form a first containing cavity and a second containing cavity, the lower shell 130 is provided with a first wall body 131, the upper cover 140 is provided with a second wall body 141, the first containing cavity 130 and the second containing cavity are separated by the butt joint of the first wall body 131 and the second wall body 141, the test paper 200 is installed in the first containing cavity, the sampling window 110, the display window 120, the detection mark T and the quality control mark C are arranged on the upper cover 140, the second containing cavity is provided with the sampling needle 300, a port 310 for taking and placing the sampling 300 needle is formed between the rear end of the upper cover 140 and the rear end of the lower shell 130, the taking and placing port 310 is provided with a mounting block 320, and the tail of the sampling needle 300 is fixed to the mounting block 320.
Preferably, the sampling window 110 is recessed inwards to form a conical sampling cavity 111, and a hole 112 is formed in the bottom wall of the sampling cavity 11 for the blood sample in the cavity to drip onto the sample pad 220.
Preferably, the sampling window 110 and the display window 120 are covered with a thin film.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the test paper comprises a PVC plate, a sample pad, a colloidal gold pad, an NC membrane and water absorption filter paper, wherein the sample pad, the colloidal gold pad, the NC membrane and the water absorption filter paper are positioned on the PVC plate and are sequentially butted from back to front, so that the test effect is more obvious, and the test is more accurate and faster; the conical sampling cavity can effectively stabilize the blood sample of a tester during detection, so that the blood sample is not easy to diffuse outwards; the second holds the chamber, and the intracavity is held at the second to the sampling needle, simple structure, and the sampling needle of being convenient for is taken along with the usefulness.
The research and development of the test paper specifically comprises the following steps:
1. novel coronavirus multi-epitope recombinant antigen analysis
Selecting spike glycoprotein, small envelope glycoprotein and membrane glycoprotein as main research objects, comparing the antigen sequence difference of the novel coronavirus with other coronavirus by adopting bioinformatics software, and simulating the spatial conformation of the antigen.
Further researching the antigen structure and the amino acid sequence in the beginning of 2 months in 2020, the inventor finds that S, E, M has dominant antigen epitope and good antigenicity and hydrophilicity, and lays a good foundation for developing a novel coronavirus IgM/IgG antibody rapid detection reagent. (the novel coronavirus surface S, E, M antigens are distributed on the surfaces of virus particles, an immune reaction is generated early in the process of infecting organisms by viruses, and the generated specific IgM/IgG antibody can be used as a novel coronavirus detection index.)
2. Novel coronavirus multi-epitope recombinant antigen tandem expression and purification
Firing 60nm colloidal gold particles according to a process formula, wherein the concentration of the colloidal gold particles is four parts per million, and the ratio of the colloidal gold particles to the gold particles is 10 ug: 1ml of colloidal gold is used for labeling the multi-epitope recombinant antigen proportionally.
2.1S, E, M antigen dominant epitope tandem
In order to enhance the recognition capability of the recombinant antigen to the novel coronavirus antibody, S, E, M antigen dominant epitopes are connected through a flexible fragment (GlyGlyGlyGlySer), and a His label is added at the carbon end of the sequence to obtain the amino acid sequence of the recombinant antigen.
2.2 optimization of nucleotide sequences encoding recombinant antigens
In order to improve the expression quantity of the recombinant antigen, on the premise that the amino acid sequence of the recombinant antigen is not changed, the amino acid sequence of the coded recombinant antigen is converted into a corresponding nucleotide sequence according to the CHO cell preferred codon, and the nucleotide sequences corresponding to the EcoRI and BamHI enzyme cutting sites are respectively added at the upstream and the downstream of the nucleotide sequence, and then the nucleotide sequence is synthesized by Hangzhou sago to Biotechnology GmbH. The synthesized target gene is cloned in pMD19-T vector (Takara Bio-engineering Co., Ltd.).
2.3 construction of recombinant antigen expression vectors
The pMD19-T vector containing the target gene and the pTT5 vector were digested with restriction enzymes EcoRI and BamHI (Bao bioengineering Daizian Co., Ltd.) at 37 ℃ for 12 hours, the digested products were subjected to 1% agarose gel electrophoresis, and the target gene and the pTT5 vector were recovered by gel cutting (gel recovery kits used in the present invention are all from Ningbo Zhongding Biotechnology Co., Ltd.). After connecting the recovered target gene and pTT5 vector at 4 ℃ for 12 hours by using T4 ligase (Baozoigaojii, Inc.), the ligation product is transformed into DH5 alpha competent cells (from Hangzhou xian to Biotechnology, Inc.), and spread on LB plate containing ampicillin resistance (50 mug/mL), after 12 hours of constant temperature culture at 37 ℃, single clone strains are picked on the plate to LB liquid culture medium containing ampicillin resistance (50 mug/mL), after 12 hours of constant temperature shaking culture at 37 ℃, plasmids are extracted by using a plasmid purification kit, and after EcoRI and BamHI double enzyme digestion identification, correct recombinant expression vector is obtained.
2.4 transfection of eukaryotic animal cells with recombinant expression vectors and purification
The constructed recombinant expression vector was transfected into CHO-K1 cells. CHO-K1 cells were passaged at a density of 1X 106/ml the day before transfection to ensure cell viability at the time of transfection, and were transfected with cell density adjusted to 2X 106/ml the day of transfection. 3.2ug of recombinant expression vector was added per ml of transfection system, and 4.8ug of PEI (Polyscience) transfection reagent was added per ml of transfection system, and the mixture was shaken up while adding. After shaking culture at 37 ℃ for 4 hours in 6% carbon dioxide, 1% 500mM VPA (sigma) and 1% 30g/L L-cysteine hydrochloride (Solebao Biotech Co., Ltd.) were added, and after shaking culture at 32 ℃ for 6 days in 6% carbon dioxide, the supernatant was centrifuged and collected, passed through a nickel agarose affinity chromatography column (Hezhou Tiandi and Biotech Co., Ltd.), the protein was removed from 20mM imidazole solution, the target protein was eluted from 300mM imidazole solution, and after collecting the supernatant, it was allowed to stand at 4 ℃ for 30 minutes, transferred into a dialysis bag having a cut-off molecular weight of 10kD to 12kD, and dialyzed overnight in PBS (10 mmol/L, pH 7.4). Immediately taking out after dialysis and subpackaging, and storing at-20 ℃ for later use.
20mM imidazole preparation: imidazole 1.36g, add 10mmol/L, pH7.4 PBS solution to dissolve to 1000 mL.
300mM imidazole preparation: imidazole 10.2g, add 10mmol/L, pH7.4 PBS solution to dissolve to make volume 500 mL.
3. Establishment and optimization of test paper
According to the process formula, concentrating the colloidal gold labeled antigen, spraying the antigen on a bonding pad, and drying at 37 ℃ to form a colloidal gold pad; coating another novel coronavirus multi-epitope recombinant antigen on an NC membrane, and drying at 37 ℃; after the sample pad is soaked in the treatment solution, drying at 37 ℃; and respectively integrating the colloidal gold pad, the NC membrane, the sample pad and the filter paper on the PVC base plate according to the process formula, simultaneously preparing a sample diluent and the negative and positive samples for testing, and optimizing the process formula according to the test result.
3.1 colloidal gold particles
The mixture is fired according to the proportion of V chloroauric acid (1%)/V trisodium citrate (1%) =5:7, the chloroauric acid is added firstly, and then the trisodium citrate is added.
3.2 marking Process
3.2.1 labeling pH 1ml colloidal gold solution was added 7ul K2CO3 (0.2M).
3.2.2 the amount of label was 16. mu.g/ml, i.e.16 ug of antigen per ml of colloidal gold.
3.2.3 labeled antigen stabilizers BSA (Roche) was chosen at a final concentration of 1%.
3.2.4 the reaction time of the labeled antigen is more than 1h, and the reaction time of the stabilizer is more than 30 min.
3.3 centrifugal purification
Centrifuging at 15 deg.C and 10000rpm for 20min, discarding supernatant, and dissolving precipitate with storage solution.
3.4 stock solutions
0.02M Tris + 15% sucrose + 5% trehalose + 1% BSA (prolitant), pH adjusted to 8.5 with hydrochloric acid.
3.5 preparation process of gold pad
3.5.1 the concentration ratio of the colloidal gold complex is 100 mul/ml, namely 1ml of the gold-labeled antigen complex, after centrifugal purification, the precipitate is redissolved by 100ul of stock solution for later use.
3.5.2 gold pad adopts Z80, width is 10mm
3.5.3 gold pad was pretreated with 1% surfactant # 1, and 5mm 30cm format was treated with 1.125ml of treatment solution
3.5.4 the gold coating amount of the colloidal gold complex was 10. mu.l/cm.
3.5.5 drying the gold pad at 37 ℃.
3.6 preparation process of cellulose nitrate membrane coated with cellulose nitrate
3.6.1 NC Membrane selection Sidolide CN 140.
3.6.2 coating detection line (T line) Process
3.6.2.1 dilution of coated antibody was 0.01M PB (7.4).
3.6.2.2 the concentration of the coated antibody is 1mg/ml of mouse anti-human IgM, 0.8mg/ml of mouse anti-human IgG and 1mg/ml of goat anti-mouse IgG.
The ejection amount of the detection line 3.6.3 was fixed at 1. mu.l/cm.
3.7 sample pad
8955 can be used as the sample pad.
3.8 sample requirement
20ul (one drop) of the sample was added and 50ul (two drops) of buffer was added.
3.9 the reaction time is 10-15 min.
After the test paper is finished, the specific detection principle is as follows: the specific antibody in the whole blood/serum/plasma of the novel coronavirus patient is combined with the multi-epitope recombinant antigen colloidal gold marker on the colloidal gold pad to form an antibody-antigen complex. When the complex flows to an NC membrane detection line (T line), the complex is combined with another novel coronavirus multi-epitope recombinant antigen to form a more complex antigen-antibody-antigen complex and gradually agglutinates into a macroscopic detection line (T line), and the unbound multi-epitope recombinant antigen colloidal gold marker flows through the T line to be captured by the antibody on the quality control line (C line) and forms a macroscopic C line. The detection results are displayed by C line and T line.
The detection reagent is used for developing a matched rapid detection system by combining technologies such as large-particle, high-concentration colloidal gold particle labeling, immunochromatography and the like on the basis of autonomously developing a novel coronavirus multi-epitope recombinant antigen, and is mainly used for on-site new rapid detection and identification of a novel coronavirus suspected patient, the expected detection time is only about 10min, and the sensitivity and the specificity are more than 95%.
The main innovation points are as follows:
the innovation point is as follows: the novel coronavirus recombinant antigen is expressed by adopting a multi-epitope tandem mode, and the detection sensitivity can be greatly improved on the premise of meeting the specificity.
In earlier work, a novel coronavirus gene sequence is deeply analyzed, bioinformatics software is adopted to simulate antigen space conformation, a plurality of different epitopes with good activity and specificity are selected, multiple tandem connection modes are tried to simulate again, the optimal epitope tandem connection scheme is selected finally, on the premise of meeting the specificity, the detection sensitivity is greatly improved, the chronic diseases with high false positive of a natural antigen are avoided, and the defect of low sensitivity of a single antigen is overcome.
The innovation point is two: optimizing the technological parameters of the immunochromatography system, improving the immunoreaction activity and greatly shortening the time required by detection.
After the novel coronavirus epidemic situation rapidly explodes, the number of the tested samples is large, the workload is large, and the operation time of operators is shortened as much as possible from the perspective of epidemic situation prevention and control. Therefore, on the premise of ensuring that the detection sensitivity and specificity are not influenced, how to improve the immune reaction efficiency to shorten the detection time is very important. The project directly detects the whole blood sample, adds unique and effective protective agent components in the formula of the sample pad treatment solution, further optimizes the technological parameters of the immunochromatography system, eliminates the negative influence of red blood cells on the premise of not influencing the detection sensitivity, and finally shortens the detection time to about 10 minutes.
The implementation of the project is not only beneficial supplement of novel coronavirus nucleic acid molecular diagnosis products, but also can provide simple, convenient and quick detection products for early screening of novel coronaviruses of basic-level medical machines, is favorable for epidemic situation prevention and control, and has higher social benefit and economic value.
Claims (5)
1. A novel coronavirus specific antibody detection kit is characterized in that: including box body and test paper, the test paper is installed in the box body, the test paper is including the PVC board, be located the PVC board and by sample pad, colloidal gold pad, NC membrane and the filter paper that absorbs water of backward preceding butt joint in proper order, and the epimembranal detection line T and quality control line C that is equipped with of NC, be equipped with the sampling window that corresponds the sample pad on the box body and correspond the display window of NC membrane, on the box body and be equipped with respectively with detection line T and the detection sign T and the quality control sign C that quality control line C corresponds at the display window side.
2. The novel coronavirus-specific antibody detection kit according to claim 1, wherein: the front end of the sample pad presses the back end of the colloidal gold pad on the PVC board, the front end of the colloidal gold pad presses the NC membrane on the PVC board, and the back end of the water absorption filter paper presses the front end of the NC membrane on the PVC board.
3. The novel coronavirus specific antibody detection kit according to claim 1 or 2, wherein the kit body comprises a lower shell and an upper cover which are connected with each other to form a first containing cavity and a second containing cavity, the lower shell is provided with a first wall body, the upper cover is provided with a second wall body, the first wall body is in butt joint with the second wall body to separate the first containing cavity from the second containing cavity, the test paper is arranged in the first containing cavity, the sampling window, the display window, the detection mark T and the quality control mark C are arranged on the upper cover, the second containing cavity is provided with a sampling needle, a taking and placing opening for taking and placing the sampling needle is formed between the rear end of the upper cover and the rear end of the lower shell, the taking and placing opening is provided with a mounting block, and the tail part of the sampling needle is fixed with the mounting block.
4. The novel coronavirus-specific antibody detection kit according to claim 3, wherein the kit comprises: the sampling window inwards caves in to form a conical sampling cavity, and a hole for blood sample in the cavity to be dripped into the sample pad is formed in the bottom wall of the sampling cavity.
5. The novel coronavirus-specific antibody detection kit according to claim 4, wherein the kit comprises: the sampling window and the display window are covered with films.
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| CN114685628A (en) * | 2022-03-24 | 2022-07-01 | 中国人民解放军陆军军医大学 | Epitope peptide of RBD of SARS-CoV-2 and its application |
| CN114685628B (en) * | 2022-03-24 | 2023-06-06 | 中国人民解放军陆军军医大学 | Antigen epitope peptide of RBD of SARS-CoV-2 and its application |
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