Method for constructing neurons with spontaneous KSHV (Kaposi's Sarcoma-Associated Herpesvirus) lytic replication

A neuron cell, construction method technology, applied in animal cells, tumor/cancer cells, vertebrate cells, etc., can solve problems such as unclear central nervous system diseases

Inactive Publication Date: 2018-09-25
SHIHEZI UNIVERSITY
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Problems solved by technology

[0004] In recent years, studies have reported that the herpes virus family in which KSHV belongs shows neurotropic tendencies, and whether KSHV can cause central nervous system diseases is still unclear.
There are a small number of PCR-based reports analyzing the presence of KSHV infection in brain tissue samples from KSHV seropositive patients, but these reports are not sufficient to clearly demonstrate that KSHV plays a role in brain parenchymal cells

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  • Method for constructing neurons with spontaneous KSHV (Kaposi's Sarcoma-Associated Herpesvirus) lytic replication
  • Method for constructing neurons with spontaneous KSHV (Kaposi's Sarcoma-Associated Herpesvirus) lytic replication
  • Method for constructing neurons with spontaneous KSHV (Kaposi's Sarcoma-Associated Herpesvirus) lytic replication

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[0055] The technical solution of the present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

[0056] Figure 1A : cultured cells, SH-SY5Y neuroblastoma cells, Vero219 cells, HEK293T cells were cultured with DMEM medium, 10% heat-inactivated fetal bovine serum (FBS), 50mg / ml streptomycin and 50U penicillin. Among them, 6 μg / ml of puromycin was added to the Vero219 cell culture medium for selection of stably infected recombinant rKSHV.219 cells. All cells were stored at 37°C, 5% CO 2 grown together under the same conditions. Replace every 3 days. The Vero219 cell line was subcultured and inoculated with 3.5×10 6 / T75 flask cells, medium 20 ml. Cells were infected with 5 ml of baculovirus Back50 expressing KSHV Rta for 4 hours. Baculovirus was removed and fresh medium containing 1.25 mM sodium butyrate was added. After 30 hours, the butyrate-containing medium was removed and fresh medium without pu...

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Abstract

The invention discloses a method for constructing neurons with spontaneous KSHV (Kaposi's Sarcoma-Associated Herpesvirus) lytic replication. The method comprises the following steps: extracting an rKSHV.219 virus with GFP and RFP fluorescent labels from a Vero219 cell, infecting with an SH-SY5Y cell, sorting cells with red fluorescence, and detecting the cell proliferation by cell counting; extracting DNA and RNA in the cells and virus DNA in cell supernatant; detecting the copy numbers of the KSHV in cells and the supernatant, and analyzing the mRNA expression levels of latent-state LANA andlytic-state ORF26; resuscitating after refrigerating the cells for one month, detecting the cell proliferation ability once again, dyeing the freshly-sorted and resuscitated SK-RG cells after being refrigerated, and comparing the changes of eFlour670 attenuation and infection fluorescence signals. The cell proliferation ability is detected, and the change of a clone fluorescence signal formed by SK-RG is detected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing neuron cells with spontaneous KSHV cleavage and replication. Background technique [0002] Kaposi's Sarcoma-associated Herpesvirus (KSHV), also known as Human Herpesvirus-8 (HHV-8), is a gamma-herpesvirus. In 1994, Chan et al. used the representative difference analysis (RDA) method to discover the virus from the skin lesions of AIDS-related Kaposi's sarcoma (Kaposi's Sarcoma) patients. In addition to KS, KSHV can also cause primary exudative lymphatic tumor (primary effusion lymphoma, PEL) and multicentric Castleman's disease (Multicentric Castleman's disease, MCD). It is known that the cell types infected by KSHV are mainly epithelial cells, endothelial cells, B cells and macrophages. [0003] KSHV can be transmitted through sexual behavior, but its main transmission route is non-sexual transmission, such as blood transmission, organ transplantation and sa...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/6851G01N15/14G01N1/30
CPCC12N5/0693C12Q1/6851G01N1/30G01N15/14C12Q2563/159
Inventor 李冬妹曹冬冬李英徐慧玲杨丽潘泽民张蕾
Owner SHIHEZI UNIVERSITY
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