A respiratory syncytial virus polypeptide antigen and its application

Abstract

The present application provides a respiratory syncytial virus polypeptide antigen and its use in the preparation of a reagent for detecting respiratory syncytial virus, wherein the amino acid sequence of the respiratory syncytial virus polypeptide antigen is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3. The polypeptide antigen screened in the present application is easy to synthesize, with small batch differences and stability; the shorter peptide segment avoids the non-specific reaction caused by longer amino acid sequences such as full-length proteins, making the detection more accurate. The non-invasive sampling and rapid colloidal gold test strip detection method established based on this has been proven to be cheaper, more efficient, and has a similar detection accuracy compared to the commonly used clinical nucleic acid quantitative detection method, and is very suitable for grassroots and clinical rapid detection.

Description

Field of the Invention

[0001] The present application belongs to the field of immunity and biological detection. Specifically, the present application provides a respiratory syncytial virus polypeptide antigen and its application. Background Art

[0002] Respiratory syncytial virus (RSV) is an RNA virus belonging to the family Paramyxoviridae. It is one of the main viral pathogens causing lower respiratory tract infections in infants and young children worldwide. The infection rate of RSV in pneumonia patients is 22.1%, making it the most common type of virus causing pneumonia. At present, the treatment of RSV is mainly supportive and symptomatic, and there are no specific drugs or vaccines on the market. The infection caused by RSV has brought a huge public health burden to the world. In the face of the above many unfavorable factors, the harm of viral infection can only be reduced through early diagnosis and early treatment.

[0003] RSV viral genes encode 10 proteins, of which G and F are the most important viral antigens, which can induce protective immune responses. G protein is a glycosylated transmembrane protein, which is a protein with large RSV genetic and antigenic variation, and is related to the adsorption of viruses and cells. According to the antigenic differences of G protein, RSV virus can be divided into two subtypes, A / B. The B subtype is mainly prevalent in Asia, and the amino acid sequence homology of type A and type B G proteins is only 53%. F protein is also a glycoprotein that mainly mediates the fusion of viruses and cells and the formation of syncytia. It can be divided into F2 region (1-109aa), cleavage polypeptide region (110-136aa) and F1 region (137-574aa). F1 region is the functional region of F protein and contains most of the antigenic epitopes. F protein has high homology in different strains, and its amino acid similarity can reach about 89%. It can stimulate the production of serum neutralizing antibodies and respiratory mucosal secretory IgA, and is the main cross-protective antigen.

[0004] Since the symptoms caused by different respiratory pathogens are relatively similar, the diagnosis of RSV infection depends not only on clinical symptoms but also on laboratory tests. The gold standard for diagnosing viral infections is virus culture, but it takes a relatively long time and requires high experimental conditions. Serological detection of IgM antibodies can help detect recent viral infections. There are ELISA and immunofluorescence methods, but they require invasive blood collection, and the operation is relatively cumbersome and has certain limitations. Although the PCR method for detecting nucleic acids has high sensitivity, swab collection is prone to nausea and vomiting, and children and the elderly are not very receptive. It also requires special fluorescent PCR amplification equipment, which is difficult to carry out in primary medical institutions. Summary of the invention

[0005] The present invention provides an amino acid sequence of a respiratory syncytial virus polypeptide, and the polypeptide antigen has been verified to be applicable to a rapid test strip for RSV IgA antibodies. The test strip uses saliva as a test sample. When a person is infected with RSV, the respiratory mucosal barrier first shows an immune response and secretes IgA to resist the virus. The qualitative detection of saliva IgA can be used to judge the RSV infection status early.

[0006] On the one hand, the present application provides a respiratory syncytial virus polypeptide antigen, the amino acid sequence of the respiratory syncytial virus polypeptide antigen is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

[0007] Furthermore, the amino acid sequence of the respiratory syncytial virus polypeptide antigen is SEQ ID NO.2.

[0008] On the other hand, the present application provides the use of the above-mentioned respiratory syncytial virus polypeptide antigen in the preparation of a respiratory syncytial virus detection reagent.

[0009] The reagents are not limited to the test papers shown in the examples, and other kits / test papers based on antigen / antibody immune reactions are also included in the protection scope of this application.

[0010] Furthermore, the respiratory syncytial virus detection reagent is an immunochromatographic test strip.

[0011] Furthermore, the above-mentioned respiratory syncytial virus polypeptide antigen is used to prepare the detection line in the immunochromatographic test paper.

[0012] On the other hand, the present application provides a respiratory syncytial virus immunochromatographic test strip, the test strip comprising a test membrane, and a test line drawn on the test membrane using the above-mentioned respiratory syncytial virus polypeptide antigen.

[0013] Furthermore, the test paper also includes a sample pad, a marking pad, a water absorbent pad, and a back plate.

[0014] Furthermore, the detection membrane is a cellulose membrane.

[0015] Furthermore, the detection membrane also has a quality control line drawn with goat anti-mouse IgG polyclonal antibody.

[0016] Furthermore, the label pad contains colloidal gold-labeled anti-human IgA mouse monoclonal antibody.

[0017] In addition to colloidal gold labeling, other existing labeling methods in the art, such as colored microspheres or fluorescent microspheres, can also be used. Those skilled in the art are familiar with the labeling and detection methods.

[0018] The structure of the immunochromatographic test paper of the present application is not limited to the above basic structure. On the basis of the unchanged antigen / detection line material, other parts and structures of the immunochromatographic test paper can also be conventionally replaced. The materials of the back plate, membrane and pad part can be selected from various available types.

[0019] The preparation method of polypeptide antigens may be a method known in the art, including but not limited to solid phase synthesis. Since the uncoupled polypeptide is a hapten, it cannot be used directly for coating, and needs to be coupled to a larger protein molecule to become a complete antigen. In the embodiment of the present invention, coupling to KLH is taken as an example, but in the specific implementation, the polypeptide can also be coupled to other inert proteins such as BSA and OVA. The role of these proteins is to provide a carrier. The key to the coating antigen is still the specific respiratory syncytial virus amino acid sequence.

[0020] Compared with the conventional detection method, the advantages of the present invention are mainly reflected in the following points:

[0021] 1. The detection method of the present invention has low cost and high cost performance.

[0022] 2. The detection method of the present invention is non-invasive to the human body, sample collection is convenient, will not cause discomfort to the tester, will not cause invasive harm to the testee, and is easily accepted by the public.

[0023] 3. The detection method of the present invention greatly reduces the risk of exposure and infection for testers and medical staff.

[0024] 4. The test strip of the present invention is easy and quick to operate, can greatly shorten the detection time, and can observe the results with the naked eye, which is very convenient for grassroots or on-site detection and is very conducive to the early diagnosis and treatment of viral infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of test strip assembly.

[0026] Figure 2 Schematic diagram of test strip detection.

[0027] Figure 3 This is a schematic diagram of the result determination. DETAILED DESCRIPTION

[0028] Example 1 Design and synthesis of antigens

[0029] The present invention analyzed the amino acid sequences of RSV virus G and F proteins using bioinformatics software, designed 3 polypeptides, synthesized by Anhui General Biological Company and coupled to KLH vector, and purified by HPLC, with the purity of all above 98%. The amino acid sequences of the 3 polypeptides are:

[0030] P1:SKPTTKQRQNKPQNREFSVNAGVTTPNDMPITNDQKKLMSSN(SEQ ID NO.1);

[0031] P2:QILPSKPTTKQRQNKPTDIFNSKYDNKIMTSKTDIRGIIKTFSN(SEQ ID NO.2);

[0032] P3:AICKRIPNKKPGKRGIIKTFSNLEGKNLYVIINYYDPLVFPSD (SEQ ID NO.3);

[0033] Anti-human IgA mouse monoclonal antibody was purchased from Hytest (Cat.#1A1cc), and goat anti-mouse IgG polyclonal antibody was purchased from Beijing Borsi Biotechnology.

[0034] Example 2 Preparation and Use of Test Strips

[0035] Preparation of test strips:

[0036] Membrane marking: The three synthetic peptides were diluted to 2.5 mg / mL with PBS at pH 7.4, and coated on the cellulose membrane at 1 μL / cm as the detection line, i.e., the T line. The goat anti-mouse IgG polyclonal antibody was diluted to 1 mg / mL with PBS at pH 7.4 at intervals of 6 mm, and then coated on the cellulose membrane at 1 μL / cm as the quality control line, i.e., the C line.

[0037] Preparation of gold-labeled antibodies: A 1.0% gold particle solution was prepared by the trisodium citrate reduction method; 5 μL K2CO3 was added to 1 mL of gold solution to adjust the pH value, and after mixing, anti-human IgA mouse monoclonal antibody (5 μg / mL) and 10 μL 10% BSA were added, mixed, and allowed to stand for 10 minutes, and then 10 μL 10% PEG20000 was added, mixed, allowed to stand for 5 minutes, and then centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and the resuspending solution (0.01 MPB + 1% BSA + 2% sucrose) was added to resuspend to obtain the gold-labeled antibody solution.

[0038] Preparation of absorbent paper: Cut the absorbent paper into 15mm wide pieces for later use.

[0039] Preparation of gold-labeled pad: Cut glass fiber into 6 mm wide strips, dilute gold-labeled anti-human IgA mouse monoclonal antibody with diluent (0.01M PB+1% sucrose+0.5% Tween 20), mix well and spray on glass fiber at 1200 μL / strip as gold-labeled pad, dry at 40°C for use.

[0040] Sample pad preparation: glass fiber was cut into 21 mm wide strips, soaked in treatment solution (0.01 M PB + 0.5% Tween 20), and then dried at 40°C for use.

[0041] Test strip assembly: Paste the coated cellulose membrane, gold label pad, sample pad, and absorbent pad on the back plate in the required order, cut into 4mm wide strips by a strip cutter, and wrap them with a plastic card shell, so that the sample pad is exposed to the sample well position of the plastic card shell, and the quality control line and test line are exposed to the result observation hole position, and assemble into a complete test card ( Figure 1 ).

[0042] Test strip detection method

[0043] Saliva sample collection: rinse your mouth with water to clean your mouth, relax your cheeks, rub them gently for 30 seconds to produce saliva, spit saliva into the collection device containing 1mL of diluent to the 2mL mark, tighten the lid, and shake well.

[0044] Break off the top of the collection tube cap and vertically drip 3 drops (about 80uL) of the pre-treated sample into the sample well of the test strip card ( Figure 2 ), and the result will be determined in 15-20 minutes.

[0045] If the saliva contains RSV IgA antibodies, they will bind to the gold-labeled anti-human IgA monoclonal antibodies and migrate to the T line of the NC membrane, where they will be specifically captured by the RSV polypeptide antigen, forming a red strip. If there is no RSV IgA antibody in the sample, the T line will not show color, and the gold-labeled human IgA mouse monoclonal antibody will continue to migrate to the C line, where it will be captured by the goat anti-mouse antibody, forming a red strip. If there is no red strip on the C line, the result is unreliable and needs to be retested ( Figure 3 ).

[0046] Example 3 Practical Effect of Test Strips

[0047] The respiratory syncytial virus IgA antibody test strip of the present invention and the detection effect of the fluorescent PCR method

[0048] A total of 80 saliva and throat swab samples were collected, and the RSV IgA antibody test strips were used for detection and comparison with the commercial RSV nucleic acid fluorescence PCR detection kit (Da'an Gene).

[0049] The test strips assembled by three polypeptide antigens were used to test clinical samples simultaneously with the fluorescent PCR kit, and the test results were compared:

[0050] Table 1 P1 antigen streaking test strip and fluorescent PCR kit test results

[0051]

[0052] Table 2 P2 antigen streaking test strip and fluorescent PCR kit test results

[0053]

[0054]

[0055] Table 3 P3 antigen streaking test strip and fluorescent PCR kit test results

[0056]

[0057] Table 4 The conformity rate between P1 / 2 / 3 antigen stripping test strip and fluorescent PCR kit

[0058]

[0059] In summary, the respiratory syncytial virus IgA test strip assembled with P2 membrane has a good detection effect on clinical samples. Compared with the respiratory syncytial virus fluorescent PCR kit, it has a 96.2% coincidence rate and has a good detection effect. The test strip uses saliva samples, which is convenient for sampling, non-invasive, and has good detection sensitivity and specificity. It is very suitable for the rapid detection of respiratory syncytial virus.

[0060] The present invention optimizes a polypeptide antigen suitable for detecting IgA antibodies to respiratory syncytial virus, whose artificial amino acid sequence is QILPSKPTTKQRQNKPTDIFNSKYDNKIMTSKTDIRGIIKTFSN, and uses the same to prepare a test strip suitable for detecting IgA antibodies in saliva samples, which has a good detection effect and a coincidence rate with the fluorescent quantitative PCR detection result of 96.2%, and can be used for clinical detection.

Claims

1. A respiratory syncytial virus polypeptide antigen, characterized in that: The amino acid sequence of the respiratory syncytial virus polypeptide antigen is SEQ ID NO.

2.

2. Use of the respiratory syncytial virus polypeptide antigen according to claim 1 in preparing a respiratory syncytial virus detection reagent.

3. The use according to claim 2, wherein the respiratory syncytial virus detection reagent is an immunochromatographic test strip.

4. The use according to claim 3, wherein the respiratory syncytial virus polypeptide antigen is used to prepare a detection line in an immunochromatographic test paper.

5. A respiratory syncytial virus immunochromatographic test strip, characterized in that: The test paper comprises a detection membrane, on which a detection line is drawn using the respiratory syncytial virus polypeptide antigen according to claim 1.

6. The test paper according to claim 5, further comprising a sample pad, a marking pad, a water absorbent pad, and a back plate. The test paper according to claim 5 , wherein the detection membrane is a cellulose membrane.

8. The test paper according to claim 5, wherein the detection membrane also has a quality control line drawn with goat anti-mouse IgG polyclonal antibody.

9. The test paper according to claim 6, wherein the label pad contains anti-human IgA mouse monoclonal antibody labeled with colloidal gold.

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