Test strip and kit for detecting the coronavirus sars-cov-2

Abstract

The application provides a test strip and a kit for detecting a novel coronavirus SARS-CoV-2. The test strip comprises an N detection line and an S detection line, the N detection line is coated with an N protein recombinant antigen, and the S detection line is coated with an S protein recombinant antigen. The test strip can directly distinguish IgM (N IgM) antibodies against the N protein and IgM (S IgM) antibodies against the S protein, and can exclude the positive S IgM antibody caused by inoculation of a non-inactivated vaccine while detecting whether the IgM antibody is positive. The test strip can solve the problem of low detection efficiency caused by multiple detections in the prior art, and is suitable for the field of in vitro diagnosis.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, and more specifically, to a test strip and kit for detecting SARS-CoV-2. Background Technology

[0002] SARS-CoV-2, the novel coronavirus, belongs to the coronavirus family and contains a spike protein (S protein), a membrane protein (M protein), an envelope protein (E protein), and a nucleocapsid protein (N protein). The pathogenic process of SARS-CoV-2 involves the binding of the receptor-binding domain (RBD) of the viral S protein to the ACE2 receptor on the surface of human respiratory epithelial cells, initiating and mediating viral invasion and pathogenicity. Therefore, the S-RBD protein is a major target for neutralizing antibodies to block SARS-CoV-2 invasion. After infection with SARS-CoV-2, the body produces many types of antibodies, including antibodies against various antigenic determinants in the N, S, E, and M proteins.

[0003] After the novel coronavirus infects the human body, it first multiplies in the respiratory system. Therefore, infection can be determined by detecting viral nucleic acid in sputum or nasopharyngeal swabs. After a period of infection, generally 7-10 days later, the body produces specific antibodies against the virus. Immunoglobulin M (IgM) antibodies are produced in the early stages of infection, usually appearing around day 7 after viral infection, rising to a certain level and becoming positive, peaking around day 14. Subsequently, IgM antibodies begin to decline as the patient gradually recovers, and rapidly decrease and disappear (becoming negative) after the virus is cleared. A positive IgM antibody test indicates that the patient is in the early stages of infection, so immunological testing methods can detect these specific antibodies and determine the extent of viral infection.

[0004] Novel coronavirus vaccines are biological products made from components related to the novel coronavirus for preventive inoculation. Based on their characteristics, they can be classified into nucleic acid mRNA vaccines, inactivated vaccines, attenuated influenza virus vector vaccines, adenovirus vector vaccines, and recombinant subunit peptide vaccines. Inactivated COVID-19 vaccines can induce antibodies against many proteins of the novel coronavirus, including the N, S, M, and E proteins. Non-inactivated vaccines, currently including mRNA vaccines, adenovirus vaccines, and recombinant protein vaccines, all use the S-RBD (receptor-binding domain of the S protein) as an immunogen. In recipients of these non-inactivated vaccines, antibodies against the S protein can be detected, while tests for other antigens such as the N protein are negative.

[0005] For serum IgM antibody testing, a positive result in individuals vaccinated with inactivated vaccines could be due to either vaccination or viral infection. To differentiate between vaccination and infection in individuals vaccinated with inactivated vaccines (primarily those targeting S-RBD), further testing for IgM antibodies against the N protein is necessary. Therefore, a product is needed that can directly distinguish between IgM antibodies against the N protein (N IgM) and IgM antibodies against the S protein (S IgM), detecting IgM antibodies while ruling out positive S IgM antibodies resulting from inactivated vaccine vaccination. Furthermore, current serum antibody testing strips primarily use whole-protein antigens, leading to lower accuracy and a higher risk of false negatives or false positives. Therefore, developing a more efficient and accurate serum antibody test strip is of significant importance. Summary of the Invention

[0006] The main objective of this invention is to provide a test strip and kit for detecting SARS-CoV-2, in order to solve the problem of low detection efficiency caused by the need for multiple tests in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a test strip for detecting SARS-CoV-2 is provided, the test strip comprising an N detection line and an S detection line, wherein the N detection line is coated with N protein recombinant antigen and the S detection line is coated with S protein recombinant antigen.

[0008] Furthermore, the N protein recombinant antigen includes at least two of the following: PA antigen, PB antigen, or PC antigen; the amino acid sequence of the PA antigen is shown in SEQ ID NO: 1, the amino acid sequence of the PB antigen is shown in SEQ ID NO: 2, and the amino acid sequence of the PC antigen is shown in SEQ ID NO: 3.

[0009] Furthermore, the PA antigen, PB antigen, or PC antigen are linked by protein linkers to form the N protein recombinant antigen; preferably, the N protein recombinant antigen is a linear protein or a cyclic protein.

[0010] Further, the PA antigen, PB antigen, PC antigen and protein linker form a first recombinant antigen unit, and the N protein recombinant antigen includes one or more first recombinant antigen units, which are linked by protein linkers; preferably, the N protein recombinant antigen includes 1-5 first recombinant antigen units; preferably, the sequence of the N protein recombinant antigen is as shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: 23.

[0011] Furthermore, the S protein recombinant antigen includes at least two of the following: PA1 antigen, PB1 antigen, or PC1 antigen; the amino acid sequence of the PA1 antigen is shown in SEQ ID NO: 4, the amino acid sequence of the PB1 antigen is shown in SEQ ID NO: 5, and the amino acid sequence of the PC1 antigen is shown in SEQ ID NO: 6.

[0012] Furthermore, the PA1 antigen, PB1 antigen, or PC1 antigen are linked by protein linkers to form the S protein recombinant antigen; preferably, the S protein recombinant antigen is a linear protein or a cyclic protein.

[0013] Further, the PA1 antigen, PB1 antigen, PC1 antigen, and protein linker form a second recombinant antigen unit, and the S protein recombinant antigen includes one or more second recombinant antigen units, which are linked by protein linkers; preferably, the S protein recombinant antigen includes 1-5 second recombinant antigen units; preferably, the sequence of the S protein recombinant antigen is as shown in SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

[0014] Furthermore, protein linkers consist of 10 to 15 amino acids.

[0015] To achieve the above objectives, according to a second aspect of the present invention, a kit is provided comprising the above-described test strip and a sample diluent.

[0016] Further, the test sample diluent is used to dilute the test sample, and the test sample diluent includes PBS buffer and surfactant S21; preferably, the concentration of PBS buffer is 0.01M and the concentration of surfactant S21 is 0.1% w / v; more preferably, the pH of PBS buffer is 7.4, and it includes 0.2 g / L KH2PO4, 2.9 g / L Na2HPO4·12H2O, and 8 g / L NaCl.

[0017] Furthermore, the samples to be tested include serum, plasma, or whole blood.

[0018] By applying the technical solution of this invention and using the above-mentioned test strip for detecting SARS-CoV-2, by placing the two proteins on the same test strip, not only can the detection efficiency be improved, but also the use of recombinant protein has the advantage of higher detection accuracy compared to whole protein. Detailed Implementation

[0019] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0020] As mentioned in the background section, existing test strips for serum antibody detection mostly use whole-protein antigens, resulting in low detection accuracy and a high risk of missed or false detections. Furthermore, they require separate detection of different antibodies, leading to low detection efficiency. Therefore, in this application, the inventors attempted to develop a novel test strip for detecting SARS-CoV-2, coating the strip with recombinant N-protein and S-protein antigens, enabling simultaneous detection of both antibodies and improving detection accuracy using these recombinant antigens. Consequently, this application proposes a series of protective measures.

[0021] In a first typical embodiment of this application, a test strip for detecting SARS-CoV-2 is provided. The test strip includes an N test line and an S test line, wherein the N test line is coated with N protein recombinant antigen and the S test line is coated with S protein recombinant antigen.

[0022] In a preferred embodiment, the N protein recombinant antigen includes at least two of PA antigen, PB antigen, or PC antigen; the amino acid sequence of PA antigen is shown in SEQ ID NO: 1, the amino acid sequence of PB antigen is shown in SEQ ID NO: 2, and the amino acid sequence of PC antigen is shown in SEQ ID NO: 3.

[0023] The test strip for detecting SARS-CoV-2 contains a recombinant N protein antigen. This recombinant N protein antigen includes PA, PB, and PC antigens. These three antigens are dominant epitope antigens screened from the whole protein (SEQ ID NO: 9) of SARS-CoV-2 in this application. By comparing the mutation sites of existing mutant strains, the conserved epitopes that have not undergone mutation were screened, and the three antigens were linked using protein linkers to obtain the aforementioned recombinant N protein antigen. Compared to existing technologies that use whole protein to prepare test strips, this antigen has a simpler spatial structure, the spatial configurations of the three antigen fragments do not affect each other, and the dominant epitopes can be better unfolded and exposed. Compared to whole protein, the aforementioned recombinant N protein antigen can bind to N IgM antibodies in the test sample more efficiently, increasing the accuracy of detection.

[0024] Furthermore, in production, due to the simple spatial structure of this recombinant antigen, modification and folding during expression are straightforward, and correct folding is easily achieved under different expression environments and systems. In contrast, whole proteins, due to their large number of amino acids and molecular weight, require more complex modifications and folding. In actual production, because the cells expressing this protein are not the actual source of the SARS-CoV-2 virus, and because large-scale expression occurs simultaneously within cells, the protein is prone to unpredictable and erroneous modifications or coiling, thus affecting its binding ability to the antigen and consequently impacting the accuracy of the test strip.

[0025] Analysis of the mutation sites of existing COVID-19 mutant strains revealed that the three dominant epitope antigens mentioned above are all amino acid fragments in the N protein of the COVID-19 virus that are structurally stable and not prone to mutation. Test strips prepared using the above recombinant antigens can detect multiple existing COVID-19 viruses and also have a good detection effect on other possible COVID-19 mutant strains.

[0026] The test strip may also include a control line, which is coated with anti-chicken IgY antibody.

[0027] In a preferred embodiment, the PA antigen, PB antigen, or PC antigen are linked by a protein linker to form an N protein recombinant antigen; preferably, the N protein recombinant antigen is a linear protein or a cyclic protein.

[0028] In a preferred embodiment, the PA antigen, PB antigen, PC antigen, and protein linker form a first recombinant antigen unit, and the N protein recombinant antigen includes one or more first recombinant antigen units, which are linked by protein linkers; preferably, the N protein recombinant antigen includes 1-5 first recombinant antigen units; preferably, the sequence of the N protein recombinant antigen is as shown in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23.

[0029] In a preferred embodiment, the S protein recombinant antigen includes at least two of PA1 antigen, PB1 antigen, or PC1 antigen; the amino acid sequence of PA1 antigen is shown in SEQ ID NO: 4, the amino acid sequence of PB1 antigen is shown in SEQ ID NO: 5, and the amino acid sequence of PC1 antigen is shown in SEQ ID NO: 6.

[0030] In a preferred embodiment, the PA1 antigen, PB1 antigen, or PC1 antigen are linked by a protein linker to form an S protein recombinant antigen; preferably, the S protein recombinant antigen is a linear protein or a cyclic protein.

[0031] In a preferred embodiment, the PA1 antigen, PB1 antigen, PC1 antigen, and protein linker form a second recombinant antigen unit, and the S protein recombinant antigen includes one or more second recombinant antigen units, which are linked by protein linkers; preferably, the S protein recombinant antigen includes 1-5 second recombinant antigen units; preferably, the sequence of the S protein recombinant antigen is as shown in SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

[0032] Similar to the aforementioned N protein recombinant antigen, the structure of the S protein recombinant antigen is a dominant epitope antigen screened from the full protein of the SARS-CoV-2 S protein (SEQ ID NO: 10). Through the connection of protein linkers, a recombinant antigen with high binding efficiency to S IgM antibodies is formed. Test strips prepared using the aforementioned S protein recombinant antigen can more accurately detect samples and prevent missed or false detections.

[0033] The test strip has two detection lines coated with recombinant N protein antigen and recombinant S protein antigen, respectively, enabling simultaneous detection of both SIgM and N IgM antibodies in the sample. This allows for the differentiation of both antibodies in a single test. This is particularly useful for subjects who have received inactivated vaccines, as these vaccines induce SIgM antibodies, making it impossible to determine SARS-CoV-2 infection solely through SIgM antibody testing. Using the aforementioned test strip coated with both N and S protein recombinant antigens, infection status can be determined in a single test, offering high efficiency, speed, and significant application value. Furthermore, simultaneous detection of both SIgM and N IgM antibodies prevents false negatives caused by low levels of one antibody that may be difficult to detect.

[0034] In a preferred embodiment, the protein linker comprises 10 to 15 amino acids.

[0035] Protein linkers, including those commonly found in existing technologies, provide flexibility to allow the proteins on either side to perform their independent functions. Excessively long linker sequences may reduce fusion protein yield and introduce immunogenicity issues; conversely, excessively short linker sequences may cause the two proteins to be too close together, affecting the folding of their higher-order structures and leading to interference and loss of protein function. Therefore, the preferred amino acid count for protein linker sequences is 10–15, including but not limited to (GGGGS). n Or (G)n. In the above-mentioned N protein recombinant antigens, PA antigen, PB antigen, and PC antigen have their own independent spatial structures and activities, each playing its own role; therefore, their linking order can be arbitrarily combined. The structure of the N protein recombinant antigen can be a linear protein composed of two linkers or a circular protein composed of three linkers, neither affecting the activity of the recombinant antigen. The protein linkers in the recombinant antigen can be the same or different, neither affecting the structure of the recombinant antigen or its antibody binding ability. The protein linkers in the above-mentioned S protein recombinant antigen also play the same role; the S protein recombinant antigen and the N protein recombinant antigen can be the same or different.

[0036] In a second typical embodiment of this application, a kit is provided, which includes the above-described test strip and a sample diluent.

[0037] The test strips described above include an N test line and an S test line. The N test line is coated with recombinant N protein antigen, and the S test line is coated with recombinant S protein antigen. The test strips may also include a control line coated with anti-chicken IgY antibody.

[0038] In a preferred embodiment, the test sample diluent is used to dilute the test sample. The test sample diluent includes PBS buffer and surfactant S21. Preferably, the concentration of the PBS buffer is 0.01M and the concentration of surfactant S21 is 0.1% w / v. More preferably, the pH of the PBS buffer is 7.4 and includes 0.2 g / L KH2PO4, 2.9 g / L Na2HPO4·12H2O, and 8 g / L NaCl.

[0039] The test sample diluent is used to dilute the test sample and includes 0.01M pH 7.4 PBS (0.2 g / L KH2PO4, 2.9 g / L Na2HPO4·12H2O, 8 g / L NaCl) and 0.1% (w / v) surfactant S21. Surfactant S21 in the test sample diluent can improve the sensitivity of the detection.

[0040] In a preferred embodiment, the test sample diluent is used to dilute the test sample. The test sample diluent includes 0.01M pH 7.4 PBS and 0.1% w / v surfactant S21 (polyoxyethylene lauryl ether, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 9002-92-0).

[0041] In a preferred embodiment, the test strip is used to distinguish between N protein infection and S protein infection. The N protein recombinant antigen coating concentration on the test strip detection line is 1.0 mg / mL, the S protein recombinant antigen coating concentration is 1.0 mg / mL, and the anti-chicken IgY antibody coating concentration on the quality control line is 1.0 mg / mL.

[0042] In a preferred embodiment, the sample to be tested includes serum, plasma, or whole blood.

[0043] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0044] Example 1: Preparation of test strips

[0045] 1. Preparation and Acquisition of Antigens and Antibodies

[0046] The N protein recombinant antigen (including specific recognition fragments of the PA, PB, and PC antigens) was produced by Shenzhen New Industries Biomedical Engineering Co., Ltd. It was obtained using standard recombinant protein expression procedures. Specifically, the full-length sequence of the SARS-CoV-2 N protein (SEQ ID NO: 9) was retrieved from the NCBI website. Epitope analysis was performed, retaining only the amino acid sequences of the PA, PB, and PC antigen segments (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively). The three antigen segments were then linked together using standard protein adapters. The corresponding gene sequences were then queried based on the selected antigen and adapter sequences and artificially synthesized. PCR was then performed, and clones expressing the correct proteins were selected for transformation and protein expression. Finally, the corresponding recombinant proteins were purified.

[0047] The recombinant S protein antigen (including specific recognition fragments of PA1, PB1, and PC1 antigens) was produced by Shenzhen New Industries Biomedical Engineering Co., Ltd. It was obtained using standard recombinant protein expression procedures. Specifically, the full-length sequence of the SARS-CoV-2 S protein (SEQ ID NO: 10) was retrieved from the NCBI website. Epitope analysis was performed, retaining only the amino acid sequences of the PA1, PB1, and PC1 antigens (SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively). A standard protein adapter was used to link the three antigen fragments together. The corresponding gene sequences were then queried based on the selected antigen and adapter sequences and artificially synthesized. PCR was then performed, and clones expressing the correct proteins were selected for transformation and protein expression. Finally, the corresponding recombinant proteins were purified.

[0048] The expression of the complete N protein (SEQ ID NO: 9) and the complete S protein (SEQ ID NO: 10) of SARS-CoV-2 were both prepared using conventional protein expression and purification methods.

[0049] Recombinant proteins were prepared using conventional protein expression and purification methods, as shown in Table 1.

[0050] Table 1

[0051]

[0052] Taking N protein recombinant antigens (N1, N4) and S protein recombinant antigens (S1) as examples:

[0053] N1 includes specific recognition fragments of two antigens, PA and PB. Recombinant proteins of PA and PB (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) were obtained using the aforementioned methods (SEQ ID NO: 11).

[0054] N4 includes specific recognition fragments of three antigens: PA, PB, and PC. PA, PB, and PC (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively) were obtained by repeating twice using the aforementioned method to form a recombinant protein with the structure PA-PB-PC-PA-PB-PC (SEQ ID NO: 17). PA, PB, and PC are all connected by protein adapters.

[0055] S1 includes specific recognition fragments of two antigens, PA1 and PB1. The recombinant proteins of PA and PB (SEQ ID NO: 4 and SEQ ID NO: 5, respectively) were obtained using the aforementioned methods (SEQ ID NO: 12).

[0056] SEQ ID NO: 1: DDQIGYYRRATRRIRGGDGK.

[0057] SEQ ID NO: 2: DGIIWVATEGALNTPKDHIGTRNPANN.

[0058] SEQ ID NO: 3: QTQGNFGDQELIRQGTDYKH.

[0059] SEQ ID NO: 4: SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH.

[0060] SEQ ID NO: 5: EVFNATRFASVYAWNRKRISNCVADYSVLYN.

[0061] SEQ ID NO: 6: EIRASANLAATKMSECVLGQSKR.

[0062] SEQ ID NO: 7: DDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKH.

[0063] SEQ ID NO: 8: SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH GGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYN GGGGSGGGGS EIRASANLAATKMSECVLGQSKR.

[0064] SEQ ID NO: 9:

[0065] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA。

[0066] SEQ ID NO:10:

[0067]

[0068] SEQ ID NO:11:DDQIGYYRRATRRIRGGDGK GGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANN。

[0069] SEQ ID NO:12:SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH GGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYN。

[0070] SEQ ID NO:13:DDQIGYYRRATRRIRGGDGKGGGGSGGGGSQTQGNFGDQELIRQGTDYKH。

[0071] SEQ ID NO:14:SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEIRASANLAATKMSECVLGQSKR。

[0072] SEQ ID NO:15:DGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKH。

[0073] SEQ ID NO:16:EVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKR。

[0074] SEQ ID NO:17:DDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKH。

[0075] SEQ ID NO:18:SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKRGGGGSGGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKR。

[0076] SEQ ID NO:19:DDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKH。

[0077] SEQ ID NO:20:SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKRGGGGSGGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKRGGGGSGGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKR。

[0078] SEQ ID NO:21:DDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKH。

[0079] SEQ ID NO:22:SKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKRGGGGSGGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKRGGGGSGGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKRGGGGSGGGGSSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHGGGGSGGGGSEVFNATRFASVYAWNRKRISNCVADYSVLYNGGGGSGGGGSEIRASANLAATKMSECVLGQSKR。

[0080] SEQ ID NO:23:DDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKHGGGGSGGGGSDDQIGYYRRATRRIRGGDGKGGGGSGGGGSDGIIWVATEGALNTPKDHIGTRNPANNGGGGSGGGGSQTQGNFGDQELIRQGTDYKH。

[0081] SEQ ID NO: 24:

[0082] The mouse anti-human IgM antibody used in this application is from Shenzhen Shengyuan Biotechnology Co., Ltd., catalog number: M2030; the chicken IgY antibody is from Keqiao (Xiamen) Biotechnology Co., Ltd., catalog number: QTAB-0104; and the anti-chicken IgY antibody is from Keqiao (Xiamen) Biotechnology Co., Ltd., catalog number: QTAB-0203.

[0083] 2. Preparation of colloidal gold: Colloidal gold was prepared by the reduction method using trisodium citrate;

[0084] Taking the production of 1000mL colloidal gold as an example, before preparing the colloidal gold, add an appropriate amount of ultrapure water to a three-necked round-bottom flask, boil it, and then discard the boiling water. Measure 990g of ultrapure water (volume = mass ÷ theoretical density) into the three-necked round-bottom flask, accurately weigh 10mL of 1% chloroauric acid solution into the flask, and install the reflux condenser. Turn on the reflux condenser and heat at 340℃ and 300rpm with stirring until boiling (continuously producing a large number of bubbles). After boiling, add 10mL of 1% sodium citrate dihydrate solution to the three-necked round-bottom flask. Start timing after the solution changes color, and stop heating after 15 minutes. After cooling to room temperature, the colloidal gold is obtained.

[0085] 3. Preparation of immunogold:

[0086] ① Preparation of immunogold immunoassay lines (N and S lines): Take 10 mL of colloidal gold solution, add 20 μL of 0.2 mol / L potassium carbonate solution, then add 100 μL of 1 mg / mL mouse anti-human IgM antibody. Stir the mixture on a magnetic stirrer at room temperature for 10 minutes. Add 100 μL of 10% BSA solution and mix well. Block the mixture at room temperature for 30 minutes, then centrifuge at 10,000 rpm for 15 minutes at 4°C. After centrifugation, discard the supernatant and resuspend the precipitate in 2 mL of gold-labeled reconstitution solution. Store at 4°C protected from light. The gold-labeled reconstitution solution consists of 100 mmol / L pH 8.0 Tris-HCl, 3% trehalose, 3% sucrose, 0.2% sodium caseinate, 0.1% Tween-20, and 0.5% PEG6000.

[0087] ② Preparation of the quality control line immunochromatographic gold: Take 10 mL of colloidal gold solution, add 20 μL of 0.2 mol / L potassium carbonate solution, then add 100 μL of rabbit anti-chicken IgY antibody at a concentration of 1 mg / mL, and stir the mixture on a magnetic stirrer at room temperature for 10 minutes. Then add 100 μL of 10% BSA solution and mix well. Block the mixture at room temperature for 30 minutes, then centrifuge at 10,000 rpm for 15 minutes at 4°C. After centrifugation, discard the supernatant and resuspend the precipitate in 2 mL of gold-labeled reconstitution solution. Store at 4°C in the dark. The gold-labeled reconstitution solution consists of 100 mmol / L pH 8.0 Tris-HCl, 3% trehalose, 3% sucrose, 0.2% sodium caseinate, 0.1% Tween-20, and 0.5% PEG6000.

[0088] ③ Mix the prepared detection line immunogold and the quality control line immunogold at a ratio of 7:3 and set aside.

[0089] 4. Preparation of immunocolloidal gold pads:

[0090] ① Gold-labeled pad pretreatment: The gold-labeled pads were cut into 30cm × 0.68cm strips. Each gold-labeled pad was pretreated using a gold spraying and coating instrument. The spraying volume of the pretreatment solution was 20μL / cm. The pretreated gold-labeled pads were then transferred to a forced-air drying oven and dried at 50℃ for 16 hours. The pretreatment solution for the gold-labeled conjugate pads consisted of 100mmol / L pH 7.4 PBS, 3% trehalose, 0.2% sodium caseinate, 0.1% Tween-20, 0.5% PEG6000, and 0.01% blocking agent.

[0091] ② Immunogold coating: Each pretreated gold-labeled pad was coated with gold using a gold coating scribing instrument at a spray volume of 33 μL / cm. After coating, the immunogold pads were placed in a forced-air drying oven and dried at 50°C for 16 hours. After drying, the immunogold pads were transferred to an electronic dehumidifier for storage.

[0092] 5. Preparation of immune NC membrane:

[0093] ① Film application: Cut the NC film (nitrocellulose film) and paste it to the corresponding position on the PVC base plate.

[0094] ② Preparation of coating buffer: The coating buffer is 0.01M PB buffer. Using 0.01M PB buffer, prepare the N protein recombinant antigen to a concentration of 1.0 mg / mL as the N-line coating buffer; using 0.01M PB buffer, prepare the S protein recombinant antigen to a concentration of 1.0 mg / mL as the S-line coating buffer; using 0.01M PB buffer, prepare the chicken IgY antibody to a concentration of 1.0 mg / mL as the control line coating buffer.

[0095] ③ Spreading: Inject the coating solution for the detection lines (N line and S line) and the quality control line into the gold sputtering spectrometer, set the spreading volume to 1 μL / cm and the moving speed to 40 mm / s, and then perform the sputtering.

[0096] ④ Transfer the scribing NC membrane plate to a 45℃ forced-air drying oven and dry for 24 hours. After drying, transfer the NC membrane to an electronic dehumidifier for storage.

[0097] 6. Sample pad pretreatment:

[0098] Prepare the sample pad pretreatment solution. Immerse the sample pads in the pretreatment solution until fully saturated. Remove the sample pads with tweezers, drain the water, place them on a sieve, and dry them overnight in a 45℃ drying oven. After drying, cut them into 2cm*30cm pieces and collect them in a drying oven for later use. The sample pad pretreatment solution formula is: 0.3% sodium chloride, 0.5% polyvinylpyrrolidone, 0.2% Tween-20, and 1mg / mL anti-RBC antibody (purchased from Hangzhou LONGi Biotechnology Co., Ltd.).

[0099] 7. Large panel assembly:

[0100] After drying, attach the immunochromatographic membrane, sample pad, gold label pad, and absorbent pad in sequence.

[0101] 8. Slice and stack cards:

[0102] Using a programmable strip cutter, set the cutting width to 4.0 mm, place the large plate on the programmable strip cutter and cut it into strips. Place the cut test strips into the corresponding positions on the bottom cover of the plastic card and secure them. Then, fasten the top cover and press firmly before placing them in a dehumidifying box for later use. This prepares test strips containing N-line and S-line recombinant antigens coated with N-protein and S-protein recombinant antigens.

[0103] 9. Test strip preparation

[0104] 1) Repeat the above preparation process to prepare test strips containing N and S lines coated with the whole protein of SARS-CoV-2 N protein (SEQ ID NO: 7) and SARS-CoV-2 S protein (SEQ ID NO: 8).

[0105] 2) Repeat the above preparation process to prepare test strips coated with different recombinant antigens, as shown in Table 2.

[0106] Table 2

[0107] NS1 N1 PA+PB S1 PA1+PB1 NS2 N2 PA+PC S2 PA1+PC1 NS3 N3 PB+PC S3 PB1+PC1 NS4 N4 (PA+PB+PC)2 S4 (PA1+PB1+PC1)2 NS5 N5 (PA+PB+PC)3 S5 (PA1+PB1+PC1)3 NS6 N6 (PA+PB+PC)4 S6 (PA1+PB1+PC1)4 NS7 N7 (PA+PB+PC)5 S7 (PA1+PB1+PC1)5

[0108] 11. Preparation of sample diluent

[0109] The sample diluent was 0.01M PBS (0.2g / L KH2PO4, 2.9g / L Na2HPO4·12H2O; 8g / L NaCl) at pH 7.4, with 0.1% (w / v) surfactant S21.

[0110] Example 2: Validation of anti-interference and mutant strain detection rate

[0111] After the novel coronavirus antibody in the sample is added to the sample pad, it enters the gold-labeled pad through capillary action. Antibodies of different proteins react and bind with the colloidal gold-labeled secondary antibody on the gold-labeled pad. Simultaneously, due to capillary action, it continues to enter the NC membrane (nitrocellulose membrane), where chromatography begins. When the bound complex molecules pass through the corresponding detection line, they react with the antigen on that detection line to achieve capture. Antibodies that do not correspond to the antigen do not react, and the complex molecules continue to chromatize on the NC membrane. When the number of captured complex molecules on the detection line accumulates to a certain amount, the colloidal gold color will appear. The C line on the test strip captures unreacted colloidal gold particles or specifically labeled colloidal gold particles, resulting in color development.

[0112] In this application, the criteria for judging visual inspection results and interpreting software results are shown in Table 3.

[0113] Table 3

[0114]

[0115] The test strips were used to detect antibodies against the novel coronavirus, including S protein IgM antibodies, N protein IgM antibodies, SARS IgM antibodies, mixed S protein and N protein IgM antibodies, influenza A IgM antibodies, influenza B IgM antibodies, and respiratory syncytial virus IgM antibodies. In addition, the test strips were used to detect IgM antibodies against the variants Omicron, Delta, Alpha, and Beta, respectively. Three test strips were used for each antibody in parallel.

[0116] Each antibody was diluted to a concentration of 0.1 mg / mL using 0.01 M PBS (pH 7.4). 75 μL of each sample was added to the sample pad area of ​​the test strip. After reacting for 10 minutes, the colorimetric results on the test strip were visually observed, as shown in Table 4.

[0117] Table 4

[0118]

[0119]

[0120] Example 3: Sensitivity Verification of Sample Diluent

[0121] The detection sensitivity was compared between the test strip prepared according to the method in Example 1 and the control test strip that used only 0.01M PBS (pH 7.4) as the sample diluent. The same strong positive sample was tested after being serially diluted with its respective diluent. The results are shown in Table 5.

[0122] Table 5

[0123]

[0124] It is evident that adding 0.1% (m / v) of nonionic surfactant S21 to the sample diluent significantly improves the detection sensitivity compared to existing techniques that use only 0.01M PBS (pH 7.4) as the sample diluent, due to the excellent solubilizing and emulsifying properties of S21.

[0125] Example 4: Antibody test strip detection rate

[0126] Whole blood and nucleic acid samples were collected from 20 patients with acute COVID-19 infection (infection within 28 days). Tests were performed using the N-line and S-line test strips and nucleic acid kits (produced by New Industries Biopharmaceuticals, catalog number: 132101005HB) coated with recombinant N-protein and S-protein antigens, respectively. The results are shown in Table 6.

[0127] Table 6

[0128]

[0129]

[0130] The results of plasma and serum tests on the collected samples were no different from those of the whole blood test.

[0131] Example 5: Sample Differentiation Test of Antibody Test Strips

[0132] The above test strips were used to test the serum of 50 healthy individuals who had recently (7-14 days) received a non-inactivated vaccine, 50 individuals who had recently received a vaccine and were infected, and 50 healthy individuals who had not received a vaccine. The results are shown in Table 7.

[0133] Table 7

[0134]

[0135]

[0136] The above samples were tested using control test strips containing N and S lines coated with full protein, and the results are shown in Table 8.

[0137] Table 8

[0138]

[0139]

[0140] As shown in Table 8, the non-detection rate of SIgM antibodies was 8% in the uninfected group (14, 33, 42, and 47); in the infected group, the non-detection rate of SIgM antibodies and / or N IgM antibodies was 16% (8, 16, 23, 24, 27, 44, 47, and 50). Particularly in infected group 24, neither SIgM nor N IgM antibodies were detected. This indicates that the control test strips prepared using coated whole protein have weak discrimination, and the detection accuracy using either whole N or whole SI protein is low.

[0141] As shown in Table 8, the test strips prepared using the recombinant N protein antigen and the recombinant S protein antigen achieved 100% accuracy for all three population groups. They can simultaneously and accurately detect both S IgM and N IgM antibodies, effectively distinguishing between populations vaccinated with inactivated vaccines.

[0142] Example 6

[0143] Test strips coated with other recombinant proteins were used to test 50 recently infected positive samples of COVID-19. The testing method and samples were the same as in Example 5. The results are shown in Table 9.

[0144] Table 9

[0145]

[0146]

[0147] As shown in Table 9, the detection rates of test strips NS1, NS2, and NS3, which were coated with only two antigens, were all low. This is presumably because recombinant proteins using only two epitopes are highly prone to false negatives. Among test strips NS4, NS5, NS6, and NS7, NS4 had the highest detection rate, close to that of the test strips prepared using the N-protein recombinant antigen and S-protein recombinant antigen in Example 5. The detection rate of NS7 was lower than that of NS4, NS5, and NS6, possibly because the antigenic sites of this recombinant protein were masked, leading to false negatives.

[0148] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the present invention, the test strip is coated with recombinant N protein antigen and recombinant S protein antigen. Compared with the whole N protein and the whole S protein, as well as proteins that only recombinantly express two epitopes, the two recombinant proteins have improved binding ability to both S IgM antibody and N IgM antibody. The test strip prepared using the two recombinant proteins can effectively avoid false negatives and has higher detection accuracy. Therefore, a series of protection schemes of this application are proposed.

[0149] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Shenzhen New Industries Biomedical Engineering Co., Ltd. <120> Test strips and kits for detecting SARS-CoV-2 <130> PN185475SZSW <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(20) <223> PA antigen <400> 1 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys 20 <210> 2 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(27) <223> PB antigen <400> 2 Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 1 5 10 15 Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn 20 25 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(20) <223> PC antigen <400> 3 Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr 1 5 10 15 Asp Tyr Lys His 20 <210> 4 <211> 35 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(35) <223> PA1 antigen <400> 4 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His 35 <210> 5 <211> 31 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(31) <223> PB1 antigen <400> 5 Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg 1 5 10 15 Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 20 25 30 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(23) <223> PC1 antigen <400> 6 Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys 1 5 10 15 Val Leu Gly Gln Ser Lys Arg 20 <210> 7 <211> 87 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(87) <223> N protein recombinant antigen <400> 7 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly 20 25 30 Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His 35 40 45 Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly Gly 50 55 60 Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg 65 70 75 80 Gln Gly Thr Asp Tyr Lys His 85 <210> 8 <211> 109 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(109) S protein recombinant antigen <400> 8 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe 35 40 45 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile 50 55 60 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 85 90 95 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg 100 105 <210> 9 <211> 419 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 9 Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr 1 5 10 15 Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg 20 25 30 Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn 35 40 45 Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu 50 55 60 Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro 65 70 75 80 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 85 90 95 Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr 100 105 110 Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln 145 150 155 160 Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser 165 170 175 Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn 180 185 190 Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala 195 200 205 Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu 210 215 220 Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln 225 230 235 240 Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys 245 250 255 Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln 260 265 270 Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp 275 280 285 Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile 290 295 300 Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile 305 310 315 320 Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala 325 330 335 Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu 340 345 350 Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro 355 360 365 Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln 370 375 380 Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu 385 390 395 400 Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser 405 410 415 Thr Gln Ala <210> 10 <211> 1201 <212> PRT <213> Severe acute respiratory syndrome coronavirus 2 <400> 10 Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr 1 5 10 15 Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg 20 25 30 Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser 35 40 45 Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr 50 55 60 Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe 65 70 75 80 Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr 85 90 95 Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr 100 105 110 Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe 115 120 125 Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu 130 135 140 Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser 145 150 155 160 Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn 165 170 175 Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr 180 185 190 Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe 195 200 205 Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr 210 215 220 Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly 225 230 235 240 Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly 245 250 255 Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr 260 265 270 Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys 275 280 285 Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser 290 295 300 Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile 305 310 315 320 Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala 325 330 335 Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp 340 345 350 Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr 355 360 365 Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr 370 375 380 Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro 385 390 395 400 Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp 405 410 415 Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys 420 425 430 Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn 435 440 445 Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly 450 455 460 Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu 465 470 475 480 Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr 485 490 495 Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val 500 505 510 Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn 515 520 525 Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn 530 535 540 Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr 545 550 555 560 Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr 565 570 575 Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr 580 585 590 Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val 595 600 605 Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr 610 615 620 Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly 625 630 635 640 Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala 645 650 655 Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala 660 665 670 Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly 675 680 685 Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr 690 695 700 Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr 705 710 715 720 Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu 725 730 735 Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn 740 745 750 Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu 755 760 765 Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp 770 775 780 Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro 785 790 795 800 Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu 805 810 815 Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile 820 825 830 Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val 835 840 845 Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala 850 855 860 Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala 865 870 875 880 Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly 885 890 895 Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala 900 905 910 Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser 915 920 925 Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala 930 935 940 Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala 945 950 955 960 Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu 965 970 975 Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu 980 985 990 Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala 995 1000 1005 Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln 1010 1015 1020 Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe 1025 1030 1035 1040 Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1045 1050 1055 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp 1060 1065 1070 Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr 1075 1080 1085 His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr 1090 1095 1100 Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile 1105 1110 1115 1120 Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe 1125 1130 1135 Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val 1140 1145 1150 Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln 1155 1160 1165 Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser 1170 1175 1180 Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp 1185 1190 1195 1200 Pro <210> 11 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(57) <223> N1 <400> 11 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly 20 25 30 Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His 35 40 45 Ile Gly Thr Arg Asn Pro Ala Asn Asn 50 55 <210> 12 <211> 76 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(76) <223> S1 <400> 12 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe 35 40 45 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile 50 55 60 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 65 70 75 <210> 13 <211> 50 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(50) <223> N2 <400> 13 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr 20 25 30 Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr 35 40 45 Lys His 50 <210> 14 <211> 68 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(68) <223> S2 <400> 14 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg 35 40 45 Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly 50 55 60 Gln Ser Lys Arg 65 <210> 15 <211> 57 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(57) <223> N3 <400> 15 Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 1 5 10 15 Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser 20 25 30 Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu 35 40 45 Ile Arg Gln Gly Thr Asp Tyr Lys His 50 55 <210> 16 <211> 64 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(64) <223> S3 <400> 16 Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg 1 5 10 15 Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn 35 40 45 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg 50 55 60 <210> 17 <211> 184 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(184) <223> N4 <400> 17 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly 20 25 30 Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His 35 40 45 Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly Gly 50 55 60 Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg 65 70 75 80 Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg 100 105 110 Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile 165 170 175 Arg Gln Gly Thr Asp Tyr Lys His 180 <210> 18 <211> 228 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(228) <223> S4 <400> 18 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe 35 40 45 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile 50 55 60 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 85 90 95 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val 115 120 125 Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys 130 135 140 Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 165 170 175 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 180 185 190 Leu Tyr Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg 195 200 205 Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly 210 215 220 Gln Ser Lys Arg 225 <210> 19 <211> 281 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(281) <223> N5 <400> 19 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly 20 25 30 Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His 35 40 45 Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly Gly 50 55 60 Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg 65 70 75 80 Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg 100 105 110 Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile 165 170 175 Arg Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly 180 185 190 Gly Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile 195 200 205 Arg Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 225 230 235 240 Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu 260 265 270 Ile Arg Gln Gly Thr Asp Tyr Lys His 275 280 <210> 20 <211> 347 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(347) <223> S5 <400> 20 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe 35 40 45 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile 50 55 60 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 85 90 95 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val 115 120 125 Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys 130 135 140 Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 165 170 175 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 180 185 190 Leu Tyr Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg 195 200 205 Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly 210 215 220 Gln Ser Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Lys 225 230 235 240 Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys 245 250 255 Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr 260 265 270 His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe Asn Ala 275 280 285 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 290 295 300 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly Ser Gly 305 310 315 320 Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys 325 330 335 Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg 340 345 <210> 21 <211> 378 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(378) <223> N6 <400> 21 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly 20 25 30 Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His 35 40 45 Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly Gly 50 55 60 Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg 65 70 75 80 Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg 100 105 110 Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile 165 170 175 Arg Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly 180 185 190 Gly Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile 195 200 205 Arg Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 225 230 235 240 Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu 260 265 270 Ile Arg Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg 290 295 300 Ile Arg Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro 325 330 335 Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly 340 345 350 Ser Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu 355 360 365 Leu Ile Arg Gln Gly Thr Asp Tyr Lys His 370 375 <210> 22 <211> 466 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(466) <223> S6 <400> 22 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe 35 40 45 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile 50 55 60 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 85 90 95 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val 115 120 125 Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys 130 135 140 Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 165 170 175 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 180 185 190 Leu Tyr Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg 195 200 205 Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly 210 215 220 Gln Ser Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Lys 225 230 235 240 Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys 245 250 255 Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr 260 265 270 His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe Asn Ala 275 280 285 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 290 295 300 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly Ser Gly 305 310 315 320 Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys 325 330 335 Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Gly Gly Gly Gly Ser 340 345 350 Gly Gly Gly Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn 355 360 365 Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp 370 375 380 Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly 385 390 395 400 Gly Ser Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 405 410 415 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 420 425 430 Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser 435 440 445 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 450 455 460 Lys Arg 465 <210> 23 <211> 475 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(475) <223> N7 <400> 23 Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly 1 5 10 15 Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Gly 20 25 30 Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His 35 40 45 Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly Gly 50 55 60 Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg 65 70 75 80 Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg 100 105 110 Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp 130 135 140 His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile 165 170 175 Arg Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly Gly 180 185 190 Gly Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile 195 200 205 Arg Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 225 230 235 240 Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu 260 265 270 Ile Arg Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly Gly 275 280 285 Gly Gly Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg 290 295 300 Ile Arg Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 305 310 315 320 Ser Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro 325 330 335 Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly Gly 340 345 350 Ser Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu 355 360 365 Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Gly Gly Gly Gly Ser Gly 370 375 380 Gly Gly Gly Ser Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg 385 390 395 400 Arg Ile Arg Gly Gly Asp Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly 405 410 415 Gly Ser Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr 420 425 430 Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gln Thr Gln Gly Asn Phe Gly Asp Gln 450 455 460 Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His 465 470 475 <210> 24 <211> 585 <212> PRT <213> Artificial Sequence <220> <221> VARIANT <222> (1)..(585) <223> S7 <400> 24 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 1 5 10 15 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 20 25 30 Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe 35 40 45 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile 50 55 60 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly 65 70 75 80 Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala 85 90 95 Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val 115 120 125 Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys 130 135 140 Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 165 170 175 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 180 185 190 Leu Tyr Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg 195 200 205 Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly 210 215 220 Gln Ser Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Lys 225 230 235 240 Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys 245 250 255 Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr 260 265 270 His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe Asn Ala 275 280 285 Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn 290 295 300 Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly Ser Gly 305 310 315 320 Gly Gly Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys 325 330 335 Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Gly Gly Gly Gly Ser 340 345 350 Gly Gly Gly Gly Ser Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn 355 360 365 Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp 370 375 380 Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Gly Gly Gly 385 390 395 400 Gly Ser Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 405 410 415 Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr 420 425 430 Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Arg Ala Ser 435 440 445 Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser 450 455 460 Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Lys Thr Gln 465 470 475 480 Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys 485 490 495 Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Gly 500 505 510 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Phe Asn Ala Thr Arg 515 520 525 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 530 535 540 Ala Asp Tyr Ser Val Leu Tyr Asn Gly Gly Gly Gly Ser Gly Gly Gly 545 550 555 560 Gly Ser Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser 565 570 575 Glu Cys Val Leu Gly Gln Ser Lys Arg 580 585

Claims

1. A test strip for detecting SARS-CoV-2, characterized in that, The test strip includes an N detection line and an S detection line, wherein the N detection line is coated with N protein recombinant antigen and the S detection line is coated with S protein recombinant antigen; The sequence of the N protein recombinant antigen is shown in SEQ ID NO: 17; The sequence of the S protein recombinant antigen is shown in SEQ ID NO:

18.

2. A reagent kit, characterized in that, The kit includes the test strip as described in claim 1 and a sample diluent.

3. The reagent kit according to claim 2, characterized in that, The sample diluent is used to dilute the sample to be tested; the sample diluent includes PBS buffer and surfactant S21, namely polyoxyethylene lauryl ether.

4. The reagent kit according to claim 3, characterized in that, The concentration of the PBS buffer is 0.01M, and the concentration of the surfactant S21 is 0.1% w / v.

5. The reagent kit according to claim 4, characterized in that, The PBS buffer has a pH of 7.4 and contains 0.2 g / L KH2PO4, 2.9 g / L Na2HPO4·12H2O, and 8 g / L NaCl.

6. The reagent kit according to claim 3, characterized in that, The sample to be tested is selected from serum, plasma or whole blood.

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