Use of Hepatocyte Growth Regulator Tyrosine Kinase Substrate in Prevention and/or Treatment of EV71 Infection

A growth regulator, tyrosine kinase technology, applied in medical preparations containing active ingredients, peptide/protein components, antiviral agents, etc.

Active Publication Date: 2017-10-03
浙江安维珞诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the preventive and therapeutic effects of HGS on the current popular and highly pathogenic enterovirus type 71 (EV71).

Method used

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  • Use of Hepatocyte Growth Regulator Tyrosine Kinase Substrate in Prevention and/or Treatment of EV71 Infection
  • Use of Hepatocyte Growth Regulator Tyrosine Kinase Substrate in Prevention and/or Treatment of EV71 Infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of hepatocyte growth regulator factor tyrosine kinase substrate

[0024] (1) Cloning of the hepatocyte growth regulator tyrosine kinase substrate gene: using the total mRNA extracted from human blood leukocytes as a template, the total cDNA was first amplified by reverse transcription PCR, and then the cDNA was used as a template to use specific The primers amplified the complete HGS gene fragment. Primers are as follows:

[0025] P1 (forward primer): 5'-AA GGATCC ATGGGGCGAGGCAGCGG-3', BamHI,

[0026] P2 (reverse primer): 5'-AA CTCGAG TCAGTCGAATGAAATGA-3', XhoI.

[0027] The HGS gene was cloned under the pTAC promoter of the vector pGEX-6p-1 by restriction digestion and ligation of BamHI and XhoI to obtain the expression vector pGEX-6p-1-HGS of HGS.

[0028] (2) Acquisition of Escherichia coli expressing hepatocyte growth regulator factor tyrosine kinase substrate: the above expression vector pGEX-6p-1-HGS was transformed into Escherichia co...

Embodiment 2

[0031] Example 2 Detection of toxicity of hepatocyte growth regulator factor tyrosine kinase substrate to host cells

[0032] After 24-48 hours of culturing human malignant embryonic rhabdomyosarcoma cells (rhabdomyosarcoma, RD) grow into a single layer (MEM medium + 10% fetal bovine serum culture), discard the culture medium, add trypsin to digest, transfer to 96-well sterile In a cell culture plate, 100 μL per well, placed at 37°C, 5% CO 2 Culture in the cell incubator for 18-24h to make the cells grow into a single layer, discard the supernatant. The HGS cryopreservation solution obtained in Example 1 was diluted step by step with serum-free MEM cell culture medium, and prepared into four concentration gradients of 8, 40, 200, and 1000 μg / mL, and then added different concentrations of HGS protein solution and discarded In the cell culture wells of the supernatant, 100 μL per well, repeat 3 wells for each concentration, and set up cell control wells (without adding drugs, o...

Embodiment 3

[0036] Example 3 Anti-EV71 virus effect of hepatocyte growth regulator factor tyrosine kinase substrate

[0037] After 24-48 hours of culture, human malignant embryonic rhabdomyosarcoma cells (RD cells) grow into a single layer (MEM medium + 10% fetal bovine serum), discard the culture medium, add trypsin to digest, and transfer to 96-well sterile cells In the culture plate, 100 μL per well, placed at 37 ° C, 5% CO 2 Culture in the cell incubator for 18-24h to make the cells grow into a single layer, discard the supernatant. Add HGS protein solutions diluted with serum-free MEM cell culture medium to a final concentration of 0.6, 1.2, 2.5, 5, 10, and 20 μg / mL, and incubate for 24 hours and then infect with EV71 virus. The multiplicity of infection (MOI) The MOI was 5, and the samples were collected after 12 hours to detect the mRNA level and protein level of EV71VP1 by Realtime PCR and Western Blot respectively. Among them, Western Blot uses β-actin as an internal reference,...

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Abstract

The invention discloses the application of hepatocyte growth regulator tyrosine kinase substrate (HGS) in preventing and / or treating EV71 infection, and belongs to the field of antiviral drugs. The present invention detects the mRNA level and protein level of EV71 VP1 by adding HGS protein into the cells and adding EV71, and finds that the mRNA and protein levels of VP1 decrease significantly compared with those without adding HGS, and it is in a dose-dependent manner, indicating that HGS has an anti-inflammatory effect. The effect of EV71. The effect of HGS on the survival rate of EV71-infected cells was further detected, and it was found that the survival rate of cells added with HGS protein was significantly higher than that of the virus control group. Preventive and therapeutic effects. The present invention discovers the new medicinal value of HGS, which can be used for preparing anti-EV71 medicine, medicine for preventing and / or treating EV71 infection.

Description

technical field [0001] The present invention relates to the field of antiviral drugs, and relates to the new application of hepatocyte growth factor-regulated tyrosine kinase substrate (hepatocyte growth factor-regulated tyrosine kinase substrate, HGS), specifically its prevention and / or treatment of enterovirus Application in drugs for type 71 (EV71) infection. Background technique [0002] Human enterovirus 71 (EV71 for short) belongs to the genus Enterovirus of the Picornaviridae family, and is a popular virus that acts on the nervous system of children. The human enterovirus 71 strain was first isolated in 1969 from infected neurological disease cases in California, USA. Rarely, EV71 can cause a disease like polio-like permanent paralysis. [0003] The EV71 virus mainly causes herpes to develop on the hands, feet, and mouth of infants, so the disease is named Hand, Foot, and Mouth Disease (HFMD). From 1970 to the present, EV71 has exploded in the Americas, Europe and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61P31/14
Inventor 邬开朗吴建国乔智罗震刘芳金晶
Owner 浙江安维珞诊断技术有限公司
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