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Preparation method of human-infected TRIM25 gene foot and mouth disease virus inhibiting cell line and application thereof

A foot-and-mouth disease virus and gene inhibition technology are applied in the preparation of TRIM25 gene, the TRIM25 gene for inhibiting the proliferation of foot-and-mouth disease virus, and the application field of TRIM25 gene in the preparation of medicines for treating or preventing FMDV, which can solve the problem of increasing the complexity of the epidemic and the difficulty of control and preventing epidemics. Difficulty at work, etc.

Inactive Publication Date: 2012-01-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no obvious cross-protection among the subtypes of FMDV, the complexity of the epidemic situation and the difficulty of control are increased, making the epidemic prevention work very difficult

Method used

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  • Preparation method of human-infected TRIM25 gene foot and mouth disease virus inhibiting cell line and application thereof
  • Preparation method of human-infected TRIM25 gene foot and mouth disease virus inhibiting cell line and application thereof
  • Preparation method of human-infected TRIM25 gene foot and mouth disease virus inhibiting cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of a kind of human TRIM25 gene suppressing foot-and-mouth disease virus cell strain:

[0046] 1. Synthesis of human TRIM25 gene:

[0047] Design a pair of primers with reference to the human TRIM25 gene sequence (NM_00508)

[0048] pTRIM25F (5'-TTTGAATTCCACCATGGCAGAGCTGTGC-3')

[0049] pTRIM25R (5'-TTTCTCGAGTCACGTAGTAGAATCGAGACCGAG-3')

[0050] RNA from Hela cells was extracted as a template, cDNA was amplified with general primers, hTRIM25 gene was amplified with specific primers phTRIM25F and phTRIM25R, and EcoRI and XhoI were introduced at its 5' and 3' ends, respectively, to facilitate subcloning. The reaction system is: template plasmid cDNA 5.0 μL, 10×LAbuffer 5 μL, phTRIM25F and phTRIM25R primers 1.0 μL each (final concentration 10 pmol / L), LA enzyme 0.25 μL, dNTPS 1.0 μL, add ddH2O to 50 μL. Act at 94°C for 3 minutes; then act at 94°C for 30 seconds; The amplified PCR product was analyzed by 0.8% (g / v) agarose gel electrophoresis, and one line w...

Embodiment 2

[0075] A kind of cell line BHK21-hTRIM25eGFP that suppresses the TRIM25 gene of FMDV proliferation is used in the preparation treatment or prevention FMDV medicine, and its application process is:

[0076] In order to study the influence of hTRIM25 gene on the proliferation of FMDV, the cytopathic effect (CPE) of the virus-infected cells was observed at different time points after infection, and the supernatant was collected to measure the virus content (TCID 50 ).

[0077] 1. Morphological observation of cells infected with FMDV:

[0078] In order to observe the effect of hTRIM25 protein on the proliferation of FMDV, BHK21-hTRIM25eGFP and BHK-eGFP cells were infected with the same dose of FMDV (0.0125 MOI), and the cytopathic effect (CPE) caused by virus-infected cells was observed at different time points after infection (attached image 3 A). The results showed that 6h after infection, BHK-eGFP cells could be found to have obvious cell rounding and enlarged intercellular ...

Embodiment 3

[0083] A kind of hTRIM25 gene that suppresses FMDV proliferation is used in the transgenic animal such as constructing transgenic pig, and its application process is:

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Abstract

The invention discloses a preparation method of a human-infected TRIM25 gene foot and mouth disease virus inhibiting cell line and the application thereof; the method comprises the steps that: A, the obtainment of hTRIM 25 gene: a pair of primers is designed by referring to a human-infected TRIM25 gene sequence; B, the construction of an eukaryotic expression vector pCA-hTRIM25eGFP: hTRIM25 is connected into the pCA vector through EcoRI and XhoI to obtain pCA-hTRIM25, then Sall and XhoI are respectively used for digesting pEGFP-C1 and pCA-hTRIM25, the vector pEGFP-C1 and a fragment containinga promoter and hTRIM25 are recovered, and are connected with T4ligase, recombinant plasmid is extracted, and the recombinant plasmid is respectively digested and identified by EcoRI and HindIII; andC, the construction of a BHK21-hTRIM25eGFP cell line which stably expresses the hTRIM25 gene: a liposome method Lipofectamine2000 is adopted to transfect the pCA-eGFP and pCA-hTRIM25eGFPG to BHK-21 cells, the screening is carried out after the transfection, and the expression of hTRIM25 protein is identified through indirect immunofluorescence and Westernblotting to obtain BHK21-hTRIM25eGFP. The application of the line in preparing FMDV treating or preventing medicine is that: the constructed BHK21-hTRIM25eGFP cell line can stably express the hTRIM25 protein; the cell line BHK21-hTRIM25eGFP which stably expresses the hTRIM25 gene can inhibit the replication of the foot and mouth disease virus; and the hTRIM25 gene is used for constructing transgenic mouses, transgenic pigs and other transgenic animals.

Description

technical field [0001] The invention relates to the fields of virology and disease resistance breeding. More specifically, it relates to a TRIM25 gene that inhibits the proliferation of Foot-and-Mouth Disease Virus (FMDV), and also relates to a preparation method of the TRIM25 gene that inhibits the proliferation of Foot-and-Mouth Disease Virus, and also relates to a method for preparing a TRIM25 gene for treating or preventing Application in FMDV medicine. Background technique [0002] my country is the largest pig raising country in the world. Pig farming is the leading industry of my country's animal husbandry, pork accounts for 67.7% of meat, and is the most important source of animal protein for our people. Pig diseases seriously restrict the development of my country's pig industry. In recent years, major diseases such as foot-and-mouth disease and porcine reproductive and respiratory syndrome have frequently broken out, causing huge losses to my country's pig indus...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/79C12N5/10G01N33/53G01N21/64A61K35/23A61P31/14A01K67/027A61K35/22
Inventor 钱平李祥敏许晋芳刘莎莎周锐金梅林陈焕春
Owner HUAZHONG AGRI UNIV
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