Polypeptide and kit for detecting infection with human herpesvirus 8
A human herpes virus, polypeptide technology, applied in the field of immunology
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Embodiment 1
[0103] This embodiment provides a polypeptide and its application.
[0104] The design of the polypeptide: the human herpesvirus type 8 viral genome is about 170kb in full length and contains at least 81 genes. Among the six capsid proteins encoded by HHV-8, the small capsid protein encoded by ORF65 is homologous to other herpes viruses, such as human herpes simplex virus, human cytomegalovirus, varicella zoster virus, Epstein-Barr virus, etc. lowest. Therefore, the ORF65 gene recombinant protein has good immunogenicity and specificity for KSHV serological analysis. In addition, HHV-8 encodes latent associated nuclear antigen LANA and envelope protein K8.1 are also commonly used in serological detection. Therefore, based on LANA, ORF65 and K8.1, the immunogenicity of the three proteins was analyzed by DNASTAR software, and 12 polypeptides were designed and synthesized. After repeated experiments, it was surprisingly found that the polypeptide (SEQ ID NO.1-SEQ ID NO .3) The ...
Embodiment 2
[0112] This embodiment provides a kit and application thereof, the kit comprising:
[0113] (1) Pre-coated microtiter plate;
[0114] (2) PBS-T washing solution;
[0115] (3) Enzyme-labeled secondary antibody;
[0116] (4) Chromogenic solution;
[0117] (5) Termination solution;
[0118] (7) Positive serum;
[0119] (8) Negative serum.
[0120] The pre-coated ELISA plate is prepared by the following method: Dissolve the polypeptide 1, polypeptide 2, and polypeptide 3 in distilled water to a concentration of 1 mg / mL, and use carbonate with a concentration of 50 mM and a pH of 9.6 Dilute the peptide concentration to 10 μg / mL with the buffer solution, coat the 96-well ELISA plate, add 100 μL to each well, coat overnight at 4 °C, then continue to add 200 μL of 5% skimmed milk powder to each well for blocking, and place in a 37 °C incubator Incubate for 1 hour and wash 3 times with PBS-T wash solution.
[0121] The preparation method of the PBS-T washing solution that appear...
Embodiment 3
[0130] This example provides an indirect ELISA detection method for human herpesvirus 8 infection, which uses the polypeptide of Example 1 and the kit of Example 2.
[0131] This embodiment has gone through the following stages before providing the indirect ELISA detection method for human herpesvirus type 8 infection:
[0132] 1. Determination of the optimal peptide and the dilution of the sample to be tested
[0133] Dissolve the peptide synthesized in Example 1 with distilled water, the storage concentration is 1 mg / mL, dilute the peptide concentration to 20 μg / mL, 10 μg / mL, 8 μg / mL, 6μg / mL, 4μg / mL, 2μg / mL and 1μg / mL, coated 96-well ELISA plate, added 100μL to each well, coated overnight at 4°C, and then continued to add 200μL 5% skim milk powder to each well for blocking , incubated in a 37°C incubator for 1 hour, and washed 3 times with PBS-T washing solution. The positive serum and negative serum against human herpes virus type 8 infection were diluted with 5% skimmed ...
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Abstract
Description
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Application Information
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