Polypeptide and kit for detecting infection with human herpesvirus 8

A human herpes virus, polypeptide technology, applied in the field of immunology

Inactive Publication Date: 2017-06-13
THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no kit for detecting herpesvirus 8 infection in China

Method used

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  • Polypeptide and kit for detecting infection with human herpesvirus 8
  • Polypeptide and kit for detecting infection with human herpesvirus 8

Examples

Experimental program
Comparison scheme
Effect test

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

The invention provides a polypeptide derived from human herpesvirus 8, an indirect ELISA antibody detection kit developed by virtue of the polypeptide and application of the polypeptide and the indirect ELISA antibody detection kit in detection of the infection with human herpesvirus 8. Experiments prove that by utilizing the indirect ELISA antibody detection kit for detecting infection with human herpesvirus 8, a specific antibody of the human herpesvirus 8 can be qualitatively detected, and the high-throughput detection of a human serum sample can be further realized, so that the infection of the human herpesvirus 8 can be rapidly diagnosed in an early stage, and the indirect ELISA antibody detection kit has relatively high clinic-use and promotional values in the diagnosis of the infection with human herpesvirus 8, the epidemiological survey and the pathogen surveillance.

Description

technical field [0001] The invention belongs to the technical field of immunology, and specifically relates to a polypeptide and an indirect ELISA antibody detection kit for human herpes virus type 8 infection. Background technique [0002] Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), belongs to γ-2 herpesviruses. HHV-8 is a double-stranded DNA virus with a full-length viral genome of about 170 kb, including at least 81 open reading frames (open reading frame, ORF). Like other herpes viruses, the vast majority of HHV-8 exists latently in the form of episomes after infecting cells, and proliferates with cell division. The product encoded by the ORF50 gene acts as a "molecular switch" to control the transition of HHV-8 from latent to proliferative state. After being activated by it, HHV-8 proliferates in large quantities, eventually causing cell rupture and releasing progeny viruses. [0003] Human herpesvirus type 8 is an import...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03G01N33/569G01N33/543
CPCC07K14/005C12N2710/16422G01N33/543G01N33/56994G01N2333/03
Inventor 程林赵方唐娴王辉陈志伟杜飞嫦何琳
Owner THE THIRD PEOPLES HOSPITAL OF SHENZHEN
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