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Anti-HPV (human papillomavirus) medicine screening model, and construction method and application thereof

An anti-human papilloma and drug technology, applied in the field of cell biology and anti-virus, can solve the problems of lack of models, inapplicable large-scale screening of anti-viral drugs, and unclear background of HPV genome, so as to achieve the effect of increasing the proportion

Pending Publication Date: 2020-07-24
BEIJING TRI PRIME GENE PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Problems and deficiencies in the above model and its establishment process: ①The operation of collecting and isolating virus particles for clinical use is relatively cumbersome, and most of the clinical samples are accompanied by co-infection of multiple subtypes, the background of HPV genome is not clear, and the influencing factors are complicated, so it is not applicable Large-scale screening of antiviral drugs in the R&D stage
② Construction of plasmids containing genomic DNA will introduce foreign gene fragments, which cannot truly simulate the replication of natural viral DNA in cells
③Skin low-risk HPV1 can cause skin proliferative warts represented by common warts. This disease is a kind of viral skin disease caused by low-risk HPV that is very difficult to treat clinically. For a long time, there has been a lack of effective models for Screening of drugs against skin low-risk HPV infection

Method used

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  • Anti-HPV (human papillomavirus) medicine screening model, and construction method and application thereof
  • Anti-HPV (human papillomavirus) medicine screening model, and construction method and application thereof
  • Anti-HPV (human papillomavirus) medicine screening model, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 11

[0069] Example 1 Establishment of Type 1 and Type 11 HPV Inhibitor Screening Models

[0070] a. Circular HPV construction

[0071] Artificially synthesized pGS1-HPV1 plasmid (the HPV1 whole genome DNA sequence refers to NC_001356, cloned into pGS1 vector through the BamHI site), pBR322-HPV11 plasmid (the HPV11 whole genome DNA sequence refers to M14119, cloned into the pBR322 vector through the BamHI site) were purchased from ATCC , The pRP-Neo plasmid is synthesized (pRP-Neo is obtained from pRP as the starting vector, and the ccdB-cmR sequence on the starting vector is replaced with the neomycin resistance gene through Gateway technology, and the specific position of the insert fragment is 804bp-1598bp , the pRP-Neo sequence is shown in SEQ ID NO: 31). After massive amplification and extraction of the plasmids, pGS1-HPV1 and pBR322-HPV11 were digested with BamHI respectively, and the band at 7.82Kb (HPV1) or 7.93Kb (HPV11) was recovered from the gel. The linearized HPV1 or...

Embodiment 2

[0082] Example 2 Reliability Verification of Type 1 and Type 11 HPV Inhibitor Screening Models

[0083] a. Detection of free HPV amplification in HaCaT[HPV1] and HaCaT[HPV11] cells

[0084] Collect 1×10^6 HaCaT[HPV1] and HaCaT[HPV11] cells respectively, extract the genomic DNA in the cells, carry out DpnⅠ or MboⅠ enzyme digestion respectively, and carry out segmented PCR amplification of the digested products, the position of the target fragment and the sequence of the primer See Table 3.

[0085] table 3

[0086]

[0087]

[0088] The amplified product was electrophoresed in 0.8% agarose gel, and the electrophoresis pattern is shown in image 3 (A and B). Among the above amplification products, Δ1-Δ6 cover the entire genome sequences of HPV1 and HPV11 respectively, including E1 and E2 regions that are easily lost when integrated into the cell genome. It can be seen from the figure that all fragments can be amplified , indicating that the HPV genome did not integrate...

Embodiment 3

[0097] Example 3 Application of Type 1 and Type 11 HPV Inhibitor Screening Models

[0098] a. HaCaT[HPV1] and HaCaT[HPV11] cells were used to detect the anti-proliferative effect of rhIFN-α1b (recombinant human α1b type interferon)

[0099] Take HaCaT[HPV1] and HaCaT[HPV11] cells and spread them on a 96-well plate, the number of cells per well is 5000 / 100 μL, set blank group, control group, 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 In the IU / mL concentration gradient group, at three detection time points of 24h, 48h and 72h, the cells were plated for 2 hours and then intervened with the corresponding concentration of rhIFN-α1b, and the MTT detection was carried out according to the scheduled detection time. Figure 6 (A and B) The results showed that the direct antiproliferative effect of rhIFN-α1b on HaCaT[HPV1] was stronger than that of HaCaT[HPV11], and it was concentration-dependent, but not time-dependent.

[0100] b. HaCaT[HPV1] and HaCaT[HPV11] cells were used to detect ...

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Abstract

The invention provides an anti-HPV (human papillomavirus) medicine screening model, and a construction method and application thereof. The medicine screening model is a HaCaT cell (cell model I) or aHaCaT cell (cell model II), wherein the HaCaT cell (cell model I) includes HPV1 circular DNA (deoxyribonucleic acid) and carries reporter gene plasmids, and the HaCaT cell (cell model II) includes HPV11 circular DNA (deoxyribonucleic acid) and carries the reporter gene plasmids. The construction method for the cell model comprises the preparation of the HPV circular DNA, quality control detection,free HPV amplification detection and the absolute quantitative detection of a copy number. The above application is that by detecting a direct anti-proliferation function for model cells and an inhibition function for HPV genome DNA replication after a medicine is applied, medicines capable of resisting the activities of HPV1 and HPV11 can be screened. A system formed by the cell model can be used for screening treatment medicines for skin low-risk type HPV (HPV1) and mucosa low-risk type HPV (HPV11), and an application prospect is wide.

Description

technical field [0001] The invention relates to the fields of cell biology and antivirus, in particular to a drug screening model for anti-human papillomavirus and its construction method and application. Background technique [0002] Low-risk HPV infection can be clinically divided into typical lesions, subclinical HPV infection without obvious skin lesions (SPI, Subclinical HPV infection) and latent infection (LPI, Latent HPV infection), among which SPI is the main clinical manifestation of HPV infection form. SPI refers to no abnormality observed by naked eyes, positive acetowhite test, atypical hyperplasia of squamous epithelium in histopathology, koilocytes or suspicious koilocytes, and positive HPV DNA detection (viral DNA exists in a low copy state in the basal It is a clinical stage of HPV infection in the cell layer), which can persist or further develop into typical warts. It is also one of the main reasons for the recurrence of proliferative warts after treatment...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/071C12Q1/02C12Q1/70C12Q1/686C12N15/11
CPCC12N5/0629C12N2510/04C12Q1/686C12Q1/701G01N33/5008C12Q2545/114
Inventor 王召静刘金毅林福玉孙纪慧程永庆
Owner BEIJING TRI PRIME GENE PHARMA CO LTD
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