Infectious clone of human cytomegalovirus and its construction method and application

A human cytomegalovirus and infectious cloning technology, applied in the direction of viruses/bacteriophages, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the problems that the genome cannot be preserved for a long time, lost genes, and cannot replicate itself , to achieve the effect of maintaining biological activity and immune activity, simple labeling method, and high fluorescence efficiency

Active Publication Date: 2020-05-22
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These drugs can inhibit the synthesis of viral DNA, so that the virus cannot replicate in cells, thereby alleviating clinical symptoms, but they cannot completely prevent the recurrence of latent infection
[0006] Herpes virus is a kind of DNA virus with large molecular weight. The full length of its genome generally exceeds 100kb. It is very difficult to study the function and pathogenic mechanism of a single gene of the virus on the basis of such a large molecular weight. The genome itself cannot be preserved for a long time, and cannot Self-replication, not to mention a series of experimental methods such as mutation transformation of molecular biology
Moreover, the long-term subculture and amplification of the virus on a fixed cell line in vitro, such as HEL, may lose some genes related to cell tropism or toxicity on the genome, so that the original shape cannot be stably preserved for a long time

Method used

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  • Infectious clone of human cytomegalovirus and its construction method and application
  • Infectious clone of human cytomegalovirus and its construction method and application
  • Infectious clone of human cytomegalovirus and its construction method and application

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Experimental program
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Effect test

Embodiment 1

[0061] The construction method of human cytomegalovirus infectious clone comprises the following steps:

[0062] The green fluorescent protein gene was inserted into the BAC to obtain the BAC vector with GFP; using the wild-type HCMV Han virus genome as a template, the left and right homologous recombination arms were amplified by PCR, and the amplified products were purified and digested sequentially. Ligation and PCR amplification are then performed to obtain the full-length sequence of the homologous recombination arm; the full-length sequence of the homologous recombination arm is connected to the BAC vector with GFP and then transformed and linearized to obtain the full-length sequence with HCMV Linearized plasmid of Han homologous sequence; Infect HEL cells with wild-type HCMV Han virus, then use the linearized plasmid with HCMV Han homologous sequence to transfect HEL cells after infection, and extract infection after subculture HEL cells and DNA with HCMV Han-BAC recom...

Embodiment 2

[0065] The construction method of human cytomegalovirus infectious clone in this embodiment comprises the following steps:

[0066] a) Remove the BamHI site from the mini-F plasmid to obtain the pUS-F2 plasmid; remove one of the restriction enzyme sites ClaI of the pUS-F2 plasmid to obtain the pUS-F3 plasmid;

[0067] b), using the plasmid pGET007 with the BamHI site removed as a template to amplify the green fluorescent protein gene, and clone the amplified product into the pGEM-T vector to obtain the pGEM-GFP plasmid;

[0068] c), using the pGEM-GFP plasmid as a template, continue to amplify the GFP fragment with restriction enzyme sites ClaI and HindIII sites, and clone it into the pUS-F3 plasmid to obtain the pUS-F4 plasmid;

[0069] d) removing the HindIII site between the two ClaI sites of the pUS-F4 plasmid to obtain the pUS-F5 plasmid.

[0070] e), extract wild-type HCMV Han viral genome DNA;

[0071] HEL cells were infected with wild-type HCMV Han virus at a multipl...

Embodiment 3

[0096] The construction method of the infectious clone of the present embodiment is realized through the following steps, and is mainly divided into the following two parts:

[0097] 1) Construction of bacterial artificial chromosome (BAC) with green fluorescent protein gene (GFP):

[0098] (11), reconnected to pUS-F2 after transformation on the basis of the original plasmid (pMBO1374, namely pUS-F1)

[0099] First put 2μg of the original plasmid pUS-F1 into the BamHI single enzyme digestion reaction system with a total volume of 20μl, and react at 37°C for 4 hours. Then, the enzyme digestion system after the reaction was purified by agarose gel electrophoresis, and the purification steps were referred to the instructions of the kit (Omega Company, USA). Continue to digest the pUS-F1DNA digested by BamHI with S1Nuclease nuclease, the reaction system is as follows: DNA 2μg, 10×S1Buffer 2μl, S1Nuclease 20U, use ddH 2Make up to 20 μl with O, react at room temperature for 15 min...

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Abstract

The invention relates to the technical field of biology, and in particularly relates to human cytomegalovirus (HCMV) infectious clone as well as a construction method and applications of the HCMV infectious clone. The construction method comprises the following steps: inserting green fluorescent protein into BAC, thus obtaining a BAC carrier with GFP; taking a wild-type HCMV Han viral genome as a template, carrying out PCR amplification on left and right homologous recombination arms of the template, carrying out purification and digestion on the product, carrying out connection and PCR amplification, connecting the full-length sequence of the left and right homologous recombination arms with the BAC carrier with the GFP, carrying out digestion and linearized transfection on HEL cells infected with the wild-type HCMV Han viruses, carrying out subculture, and extracting the total DNA of the cells; and electro-transforming the total DNA into competent cells, and culturing in a resisting culture medium. The biological properties of the recombinant viruses generated by the infectious clone are similar to that of the wild-type viruses, and the infection condition of the infectious clone of the viral strains in the cells is observed through the expression of GFP reporter genes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human cytomegalovirus infectious clone and its construction method and application. Background technique [0002] Human cytomegalovirus (HCMV) belongs to the β subfamily of herpesviruses. Its genome is about 230kb and encodes more than 200 open reading frames (open reading frame, ORF). It is currently the largest known human herpesvirus. The transcription of HCMV genes in the host cells has a time phase, which can be divided into three categories: immediate early (IE), early (early, E) and late (late, L). Among them, viral proteins expressed by immediate early genes such as IE1 and IE2 (also known as UL123 and UL122) are the most important transcriptional activators during viral replication, and are also necessary regulatory factors for early and late gene expression; proteins encoded by early genes such as UL44 and pp65 are mainly related to gene replication; while late proteins ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/65C12N15/85C12Q1/70C12Q1/686G01N33/68G01N33/569
Inventor 罗敏华谌章舟柳中洋赵非阮强朱桦
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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