Human herpes virus hominis pyrolysis, reproduction and activation host marker and purpose thereof

A herpes virus and marker technology, applied in the field of biomedicine, can solve problems such as unified indicators of herpes virus lysis and replication activation detection

Inactive Publication Date: 2020-02-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no unified index for the detection of herpes virus cleavage and replication activation.

Method used

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  • Human herpes virus hominis pyrolysis, reproduction and activation host marker and purpose thereof
  • Human herpes virus hominis pyrolysis, reproduction and activation host marker and purpose thereof
  • Human herpes virus hominis pyrolysis, reproduction and activation host marker and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: KSHV-positive cells are activated by viral lysis and replication to down-regulate the expression of STAT6 protein

[0041] KSHV-negative cells (BJAB) and KSHV-positive cells (BCBL-1 and BC-3) were all from ATCC preservation, and the above three kinds of cells were treated with TPA (20ng / ml) and sodium butyrate NaB (3mM), at 37°C, 5 %CO 2 Humidified for 24 hours. After the cells were collected by centrifugation, they were lysed on ice with RIPA lysate for 30 min, and after centrifugation, SDS loading buffer was added and boiled for 5 min. After separation by SDS-polyacrylamide gel electrophoresis, transfer to nitrocellulose membrane, block with 5% degreaser milk powder at room temperature for 30min, and carry out anti-STAT6 (D3H4, Cell Signaling Technology), STAT3 (79D7, Cell Signaling Technology), KSHV virus respectively Western blot detection of protein RTA and cell reference protein GADPH (G8140-01, US Biological). The results showed that TPA and NaB tre...

Embodiment 2

[0052] Example 2: KSHV encodes immediate early protein RTA to degrade STAT6 protein

[0053] It is known that RTA is a key protein in KSHV transition from latent phase to cleavage replication phase, and has the function of E3 ligase, which can participate in the process of ubiquitination modification. To explore whether RTA is involved in the downregulation of STAT6. The iSLK-RTA cells were treated with 2ug / ml DOX to activate the expression of RTA, and protein samples were collected at 6 and 12 hours, respectively. The expression of STAT6, STAT3, RTA, and internal control protein GADPH was detected by Western blot. The experimental results showed that the expression level of STAT6 protein decreased with the increase of RTA protein expression, while STAT3 protein did not change significantly. It shows that KSHV RTA is involved in the degradation of STAT6 protein.

Embodiment 3

[0054] Example 3: RTA interacts with STAT6 protein

[0055] In order to further explore the relationship between RTA and STAT6 protein, we constructed expression plasmids RTA-myc and FLAG-STAT6 carrying different tags respectively. The method of transfection reagent PEI was used to transfect RTA-myc and FLAG-STAT6 into 293 cells individually or co-transfected according to the volume ratio of plasmid and PEI 1:3, and the cells were harvested after 48 hours of transfection. After centrifugation, the cells were lysed with RIPA lysate on ice for 30 min, and 5% of the supernatant was removed as input after centrifugation, and ProteinA / G+IgG was added to the remaining supernatant to remove non-specific binding. After centrifugation, take the supernatant and add anti-myc primary antibody overnight, add ProteinA / G to rotate and shake for 2h, after centrifugation, the beads are washed 4 times with RIPA lysate, add SDS protein buffer and boil for 5min. The expression of STAT6 and RTA w...

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Abstract

The invention belongs to the technical field of biomedicine, and relates to a human herpes virus hominis pyrolysis, reproduction and activation detection marker, in particular to the purpose of a signal transducer and activator of transcription 6 (Signal transducer and activator of transcription 6,STAT6) as the human herpes virus hominis pyrolysis, reproduction and activation detection marker. Target protein-STAT6 which is consistently regulated during human herpes virus pyrolysis and reproduction is obtained from the host angle, the experiment result shows that the STAT6 protein is significantly reduced in the infection course of Kaposi sarcoma herpesvirus (KSHV), and the STAT6 protein can be used as the marker for detecting pyrolysis, reproduction and activation of various herpes viruses, can be further used as a common target for diagnosing and treating herpes virus relevant tumor diseases, and can be further used for preparing products for preventing, detecting and treating human herpes virus hominis infection.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a human herpes virus lytic replication activation detection marker, in particular to signal transducer and activator of transcription 6 (Signal transducer and activator of transcription 6, STAT6) as a human herpes virus lytic replication activation detection marker the use of. [0002] The present invention also relates to the use of STAT6 protein as a marker in antiviral aspects, including the use in preparation of products for the prevention, detection and treatment of human herpes virus infection. [0003] technical background [0004] According to the World Health Organization, more than 90% of the world's population is infected with different types of herpes viruses (1) . Herpesviruses belong to the family Herpesviridae. Eight herpesviruses are currently known to infect humans, and they are divided into three subfamilies: α, β, and γ according to their infection character...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61P31/22A61P35/00G01N33/533
CPCA61K45/00A61P31/22A61P35/00G01N33/533
Inventor 蔡启良顾峰王冲朱彩霞王玉燕
Owner FUDAN UNIV
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