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Method for analyzing action mechanism of anti-herpes virus medicament
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An analytical method, a technology of drug action, applied in the fields of biochemical equipment and methods, material excitation analysis, and microbial determination/inspection, to achieve the effect of narrowing the scope, reducing the investment of time and money
Active Publication Date: 2012-12-26
广州润广弘腾信息技术有限公司
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Problems solved by technology
Usually, the mechanism of action is elucidated by looking for components that interact with drug molecules, but this will consume a lot of manpower, material resources and time due to the wide range and unclear targets
Method used
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Embodiment 1
[0045] Example 1: Construction of Vero-ICP10-Luciferase
[0046]Using HSV2333 DNA as a template, the ICP10 gene promoter sequence was amplified by primers ICP10F: 5′-CGAGATCTGTCGACAGG-CTGTACCGCTGG-3′, ICP10R: 5′-CCAAGCTTGTCGACAGGACAGCACC-GGCC-3′, and then cloned into the single clone site of pGL4.17-basic vector Point between BglII and HindIII to obtain recombinant plasmid pGL4-ICP10-Luciferase. The recombinant plasmid pGL4-ICP10-Luciferase was transfected into Vero cells by Lipfectin2000 (Invitrogen Corp.), and selected by G418 to obtain a stable monoclonal cell line Vero-ICP10-Luciferase.
Embodiment 2
[0047] Embodiment 2: the construction of recombinant virus HSV / Blue
[0048] The recombinant reporter virus HSV / Blue obtained by inserting a lacZ gene coding frame controlled by the ICP4 promoter into the TK gene (47,359-47,412) of the HSV-1 KOS strain through Sac I and Sph I restriction sites.
Embodiment 3
[0049] Embodiment 3: Analysis method based on recombinant virus
[0050] Using HSV / Blue to analyze the mechanism of drug action, the method diagram is as follows figure 1 Shown:
[0051] A. Determine whether the drug acts on the stage after HSV adsorbs cells and before virus replication.
[0052] After the Vero cell monolayer was pre-cooled at 4°C for 10 minutes, inoculated with 5 MOI of HSV / Blue, adsorbed at 4°C for 1.5 hours, discarded the virus solution, then added drugs at gradient concentrations, and cultured at 37°C for 5 hours, then discarded the culture solution. Cells were lysed for β-gal activity detection;
[0053] B. Determine whether the drug inhibits virus adsorption by interacting with cells.
[0054] The Vero cell monolayer was pre-cooled at 4°C for 10 minutes, then added drugs at gradient concentrations, incubated at 4°C for 1.5 hours, discarded the drug solution, inoculated with 5 MOI of HSV / Blue, continued to culture at 4°C for 1.5 hours, and then transf...
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Abstract
The invention discloses an analyzing method of an action mechanism of an anti herpesvirus medicament, which utilizes a recombinant virus provided with a reporter gene controlled by a virus immediate early promoter, or a reporter cell system as an analyzing system, combines different processing modes and a specific detecting time for carrying out detection and then carries out identification on the action mechanism of the anti herpesvirus medicament according to the result and an identification scheme. At the temperature of 4 DEG C, three different processing modes that the virus is firstly absorbed and then medicated, the medicament is firstly acted with cells and then the virus is added and the virus is firstly acted with the medicament and then added on the cell together are adopted; and then the mixture is transferred into the temperature of 37 DEG C and cultured for 4 to 7 hours, the activity of the reporter gene is detected and finally the result is identified. The method can judge the action step of the medicament in one day, is easy and quick, greatly shortens the range for searching the action mechanism of the anti herpesvirus medicament, reduces the investment on time and money and is an excellent tool for developing the anti herpesvirus medicament.
Description
technical field [0001] The invention relates to the field of analysis methods for drug action mechanisms, in particular to an analysis method for the action mechanism of anti-herpes virus drugs. Background technique [0002] Herpesviruses are DNA-enveloped viruses that include a variety of human and animal herpesviruses. There are currently 8 human herpes viruses, which are divided into three subfamilies α, β and γ, including human herpes simplex virus type 1 and type 2 (HSV1, HSV2), varicella-zoster virus (VZV), cytomegalovirus (CMV), herpesvirus types 6 and 7, Epstein-Barr virus, and Kaposi's sarcoma-associated virus. Human herpes virus can infect various tissues of the human body, causing infections in the skin, mucous membranes, lymphatic system, reproductive system, nervous system, liver, lungs and other organs. Herpes virus infection has the characteristics of latent, recurrent and transformed cells, which can cause a variety of diseases, congenital infection and ne...
Claims
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