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Anti-human-cytomegalovirus drug screening method and application thereof

A human cytomegalovirus and drug technology, applied in the biological field, can solve problems such as limited observation time, high detection cost, and difference in detection results, and achieve the effects of shortened detection time span, flexible detection time, and short detection cycle.

Pending Publication Date: 2018-10-12
ZHEJIANG HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main means of the traditional determination of the anti-HCMV effect of drugs are: (1) the degree of cytopathic changes (CPE) is determined, mainly through microscopic observation with the naked eye, and the condition of cytopathic changes is recorded every day, which needs to be observed for 5-10 days; (2) plaque counting In the test, plaques are caused after virus amplification, stained, and then observed and counted by a microscope to count the number of plaques formed; (3) Virus titer determination, the result is accurate, but the operation is very cumbersome; (4) Viral DNA replication and transcription product determination
These indicators can be used to detect the anti-HCMV activity of a single or a small number of drugs, but they are insufficient for screening anti-HCMV drugs: (1) There are too many manual operations (such as observation through a microscope), and there are large subjective differences in the evaluation of cell lesions. Plaque counting errors are also large, lack of objective quantitative data, and need to be observed in time, and the observation time is limited; (2) for the plaque formation test and virus titer measurement, it must wait until the HCMV replicates to the middle and late stage before detection, The cycle is long and the operation is cumbersome; (3) direct measurement of viral DNA copy number and transcription product results are accurate, but the operation steps are many, and the detection cost is also high. It can be used for the verification of anti-HCMV activity, but it is not suitable for drug screening
In addition, some researchers have constructed a HCMV-TOWNE-GFP recombinant cytomegalovirus, which can judge the anti-HCMV effect of the drug by detecting the expression of green fluorescent protein (patent publication number: CN101319202A), but in the actual operation process, there are The following problems: 1) The objectivity of the test results is insufficient. The expression of green fluorescent protein is judged by the naked eye to judge its fluorescence intensity, and there is no quantitative data given by the instrument; 2) It needs to be tested in a living cell state and must be observed in time, and the detection time is limited. , and the test results vary with the cell state; 3) Need to be equipped with an inverted fluorescence microscope
Therefore, there is still a lack of a flexible, reliable, efficient and rapid anti-HCMV drug screening method

Method used

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  • Anti-human-cytomegalovirus drug screening method and application thereof
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  • Anti-human-cytomegalovirus drug screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: HCMV infects WI-38 cells to induce an increase in mitochondrial content. The specific steps are as follows:

[0044] (1) Cell culture and HCMV inoculation method

[0045] Using 30PD human embryonic lung diploid fibroblast WI-38, press 2×10 with 10% FBS medium 4 / cm 2 Inoculate into a culture dish, replace the medium with 0.2% FBS after 24 hours and continue to culture for 48 hours. Through this method of serum starvation, the cells are synchronized at G0 / G1 at this time, which is more conducive to the infection of HCMV. Afterwards, the HCMV virus (Towne virus strain) was inoculated with an inoculation amount of 0.01 MOI (multiplicity of infection), and the culture was continued until the specified time for relevant detection.

[0046] (2) Determination of the number of mitochondria in WI-38 cells after HCMV infection

[0047] Cardiolipin (CL) in the mitochondrial inner membrane is a specific component of mitochondria. The fluorescent probe nonylacridine or...

Embodiment 2

[0048] Example 2: Resveratrol inhibits the increase of mitochondrial content in WI-38 cells after HCMV infection

[0049] Resveratrol (RES) is a drug (Antiviral Res.2004,63:85-95) with anti-HCMV activity reported in the literature. In this example, it is used as a positive drug against HCMV. is 20 μM. Cell culture and HCMV inoculation method are with embodiment 1 described, set up the control group (MOCK) that does not add HCMV, only add HCMV group (HCMV) and resveratrol test group (HCMV+RES), resveratrol 10 μ M or 20 μM was added to the medium 2 hours before HCMV inoculation, and the cells were collected 3 days after HCMV inoculation (3d.p.i.) for detection of mitochondrial content in WI-38 cells. The detection method was the same as that described in Example 1. see results figure 2 , HCMV-induced increase in mitochondrial content can be significantly inhibited by resveratrol (RES), indicating that resveratrol has good potential anti-HCMV activity in this experimental syst...

Embodiment 3

[0050] Example 3: Resveratrol significantly inhibits HCMV-related protein expression and DNA replication

[0051] The MTT test showed that the half toxic concentration (TD50) of resveratrol on WI-38 cells was about 100 μM, so its therapeutic index (TI) = TD50 / IC50>10, indicating that it had good potential anti-inflammatory effects in this experimental system. HCMV activity. In order to further confirm the anti-HCMV effect of resveratrol (RES) in this experimental system, we detected the inhibitory effect of resveratrol on the expression of HCMV immediate early protein (IE) and DNA amplification. Cell culture and HCMV inoculation method are with embodiment 1 described, set up the control group (MOCK) that does not add HCMV, only add HCMV group (HCMV) and resveratrol test group (HCMV+RES), resveratrol 20 μ M in Added to the culture medium 2 hours before HCMV inoculation, collected cells at different time points after HCMV inoculation, and performed Western Blot detection of HCM...

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Abstract

The invention relates to an anti-human-cytomegalovirus (HCMV) drug screening method and application thereof. The anti-human-cytomegalovirus (HCMV) drug screening method is characterized in that mitochondrial content in human embryonic lung fibroblasts can be induced to increase using the HCMV, a significant correlation exists between increase of the mitochondrial content and amplification of the HCMV, an inhibition ratio of drugs to the mitochondrial increment induced by the HCMV is calculated by measuring a variation of the mitochondrial content, and the anti-HCMV effect of the drugs is effectively reflected. The anti-human-cytomegalovirus (HCMV) drug screening method has the characteristic of being easy to operate, flexible in detecting, sensitive in reaction and accurate in result.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for screening anti-human cytomegalovirus drugs and its application. Background technique [0002] Human cytomegalovirus (HCMV) belongs to the β-subfamily of herpesviruses and is common in the population, especially the elderly, pregnant women and newborns with weakened immunity. It is also an important factor causing birth defects. HCMV infection can cause damage to the nervous system, liver, respiratory system and blood system, and even life-threatening in severe cases (RevMed Virol, 2010, 20:311-326). Data show that my country is a high-incidence area of ​​HCMV infection, and the anti-HCMV positive rate in children is 83.2%-87.2%, and it is as high as 95% in adults (International Journal of Laboratory Medicine, 2010, 31:1131-1133). Therefore, active and effective treatment of CMV-infected patients is an important means to improve the quality of the newborn population and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12Q1/70C12R1/93
CPCC12N2503/02C12Q1/025G01N33/5008G01N2500/10
Inventor 毛根祥严静王国付王三应苏慧丽代继桓杨舟鑫张婧暴一众
Owner ZHEJIANG HOSPITAL
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