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Method for constructing HCMV encoding miRNA deletion strain based on Crispr-cas9 technology and application thereof

A gene and target technology, applied in the field of constructing HCMV-encoded miRNA deletion strains, can solve the problems of inability to miRNA regulation, long time period, and high equipment requirements

Pending Publication Date: 2022-01-11
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, simple mimics or inhibitors of miRNAs cannot regulate HCMV-encoded miRNAs stably for a long time
However, the traditional use of artificial chromosome (BAC) technology to transform viruses with CMV technology faces more difficulties, such as high technical and equipment requirements, long time period, and low success rate. - HCMV strain method

Method used

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  • Method for constructing HCMV encoding miRNA deletion strain based on Crispr-cas9 technology and application thereof
  • Method for constructing HCMV encoding miRNA deletion strain based on Crispr-cas9 technology and application thereof
  • Method for constructing HCMV encoding miRNA deletion strain based on Crispr-cas9 technology and application thereof

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preparation example Construction

[0050] The preparation method of the LB plate with ampicillin resistance is: 1) get ampicillin (0.5g / bottle) (Yuekang Pharmaceutical Group Co., Ltd. product, batch number: 22081002), add 5mL deionized water after autoclaving, directly Mix well to obtain a stock solution of ampicillin with a concentration of 100 mg / mL, and freeze it at -20°C for later use; 2) Accurately weigh 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar powder, add deionized water to make up to 1 L, and autoclave Bacteria; after cooling to 50°C, add ampicillin stock solution at a ratio of 1:1000, mix well, quickly pour it into a high-pressure plate and let it stand, after cooling and solidifying, invert the plate to obtain an LB plate with ampicillin resistance, 4° Refrigerate and set aside.

[0051] The preparation method of the 10% fetal calf serum DMEM culture medium is: in the DMEM medium, add serum according to 10% volume under sterile conditions, the serum product number (04-001-...

Embodiment 1

[0057] The preparation of embodiment 1 △ miRNA-HCMV virus strain

[0058] For the preparation flow chart of expressing sgRNA-Cas9 lentivirus, see figure 1 ,Specific steps are as follows:

[0059] 1. Construction of vector pLenti-US33as-5p-sgRNA

[0060] 1. Design the target sequence of miR-US33as-5p

[0061] The sequence of hcmv-miR-US33as-5p is as follows:

[0062] 5'-GGAUGUGCUCGGACCGUGACGGUG-3' (as sequence 1 in the sequence listing)

[0063] In order to knock out the hcmv-miR-US33as-5p gene, select sgRNA, its name is US33as-5p-sgRNA, and its nucleotide sequence is 5'-GCUCACGUUUGGAUGUGCU-3'. The coding strand sequence of the US33as-5p-sgRNA gene is: 5'-GCTCACGTTTGGATGTGCT-3' (such as the 1st-19th position of sequence 2 in the sequence listing)

[0064] For the US33as-5p-sgRNA gene, DNA primers F and R (underlined nucleotides are reverse complementary) were designed and synthesized by Beijing Aoke Biological Company.

[0065] F: 5'-CACCG GCTCACGTTTGGATGTGCT -3';

[0...

Embodiment 2

[0105] Example 2, hcmv-miR-US33as-5p target identification and verification of its protective effect on interferon

[0106] 1. Screening and identification of hcmv-miR-US33as-5p target

[0107] 1. hcmv-miR-US33as-5p target screening

[0108] Use prediction software to predict and analyze the target of hcmv-miR-US33as-5p (also known as hcmv-US33as-5p), the results are shown in Image 6 , and the predicted targets are the 13 genes in Table 1, respectively.

[0109] Table 1 The sequences of different primer pairs designed for the predicted target of hcmv-miR-US33as-5p

[0110]

[0111]

[0112] The targets predicted by the software were respectively constructed with dual luciferase reporter plasmids containing the predicted target 3'-UTR, and co-transfected with the hcmv-miR-US33as-5p mimic into 293FT cells, and the predicted target 3'-UTR was transferred and expressed 13 kinds of luciferase recombinant 293FT cells, 293FT cells transfected with predicted target 3'-UTR+NC-R...

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Abstract

The invention provides a method for constructing recombinant human cytomegalovirus and application thereof. According to the method for constructing the recombinant human cytomegalovirus, the HCMV encoding miRNA deletion strain is rapidly constructed with high specificity by utilizing a Crispr-Cas9 technology, the obtained HCMV encoding miRNA deletion strain has gene mutation at the position encoding hcmv-miR-US33as-5p, and no sequence changes at other sites, and can be passaged in susceptible cells.. The invention also utilizes the constructed HCMV encoding miRNA deletion strain to confirm that the IFNAR1 is a target of hcmv-miR-US33as-5p, and the restoration of the host cell IFNAR1 can be up-regulated by inhibiting the expression of the miRNA molecule. Aiming at the hcmv-miR-US33as-5p, the HCMV encoding miRNA deletion strain has the value of being used as a candidate drug for preventing and treating the herpesvirus infection.

Description

technical field [0001] The invention relates to a method for constructing HCMV coding miRNA deletion strain based on Crispr-cas9 technology and its application. Background technique [0002] Cytomegalovirus (CMV) belongs to the β-herpes virus family, and its modes of transmission include blood transfusion, mother-to-child transmission, and sexual transmission, etc., and the infection rate in the population is extremely high. Studies have found that microRNA (microRNA, miRNA) encoded by CMV may serve as a non-antigen immunomodulatory means and play an important role in the latent process of the virus. At present, there are more than 230 virus-encoded miRNAs recorded in the miRBase database, most of which are encoded by herpes viruses. A total of 26 mature miRNAs encoded by human cytomegalovirus (HCMV) and 29 mature miRNAs encoded by murine cytomegalovirus (MCMV) have been identified and discovered. The miRNA functions encoded by CMV include: miR-US33-5p targets the STX3 (Sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/113A61K45/00A61P31/22C12R1/93
CPCC12N7/00C12N15/85C12N15/113A61K45/00A61P31/22C12N2310/141C12N2310/20
Inventor 张艳宇邓江宋鑫马平吕丽萍张骞张阳阳周锡鹏许金波
Owner ACADEMY OF MILITARY MEDICAL SCI
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