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Cattle Nramp1 macrophage specificity expression vector as well as construction method and application thereof

An expression vector and construction method technology, applied in the field of genetic engineering, can solve problems such as troubles in the prevention and control of intracellular parasitic bacteria, and achieve the effects of reducing adverse effects and increasing expression levels

Inactive Publication Date: 2011-10-26
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, intracellular parasitic microorganisms have a set of mechanisms to resist the killing of macrophages to prevent the body from clearing them, and grow and reproduce in macrophages, which brings great troubles to the prevention and treatment of intracellular parasitic diseases

Method used

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  • Cattle Nramp1 macrophage specificity expression vector as well as construction method and application thereof
  • Cattle Nramp1 macrophage specificity expression vector as well as construction method and application thereof
  • Cattle Nramp1 macrophage specificity expression vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Isolation, amplification and sequencing of Nramp1 gene fragments

[0045] 1.1 Extraction of total RNA

[0046] The jugular vein blood of Qinchuan cattle (Shaanxi Yangling Keyuan Bioengineering Co., Ltd.) was aseptically collected, and red blood cell lysate (Triis-NH 4 Cl), mix at 4°C for 4 to 5 minutes (during which repeated gentle inversion and mixing) to remove red blood cells, wait until the red blood cells are completely broken, centrifuge at 1000r / min for 5 minutes to obtain white blood cell pellet, wash with PBS 2 to 3 times, and centrifuge to precipitate. Add an appropriate amount of TRIzol reagent, let it stand at room temperature for 5 minutes, add 1 / 5 TRIzol reagent amount of chloroform, shake and mix, let it stand at room temperature for 5 minutes, centrifuge at 12000r / min at 4°C for 10 minutes, absorb the supernatant and transfer it to a new centrifuge tube, add an equal volume of Isopropanol, let stand at room temperature for 10min, centrifuge at...

Embodiment 2

[0065] Example 2 Construction and Identification of Nramp1 Eukaryotic Expression Vector Recombinant Plasmid pIRES2-N

[0066] The pMD18-N4 and pIRES2-EGFP vectors were digested with BamH I and Sal I respectively, and the target gene fragments recovered from the gel were ligated with the corresponding vector fragments with T4 DNA ligase overnight at 16°C, and the ligated products were transformed into DH5a competent bacteria, and after 16 hours Single clones were picked and shaken, amplified, and recombinant plasmids were extracted for enzyme digestion identification, and the identified correct recombinant plasmids were named pIRES2-N( Figure 4 ).

Embodiment 3

[0067] Example 3 Construction and identification of phagocyte-specific expression vector pIRES2-PN

[0068] 3.1 Blood genome extraction

[0069] (1) Take 200 uL of peripheral blood samples from Holstein cows (Shaanxi Yangling Keyuan Bioengineering Co., Ltd.), add 20 uL of proteinase K solution, and mix well. Add 200uL of buffer GB again, mix thoroughly by inversion, and place at 70°C for 10 minutes. After the solution becomes clear, centrifuge immediately to remove the water droplets on the inner wall of the tube cap.

[0070] (2) Add 200uL absolute ethanol, shake fully for 15s, and centrifuge briefly.

[0071] (3) Add the solution and flocculent precipitate obtained in the previous step into the adsorption column CB3, centrifuge at 13400×g for 30 s, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0072] (4) Add 500uL buffer GD to the adsorption column CB3, centrifuge at 13400×g for 30s, discard the waste liquid, and put the adsor...

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Abstract

The invention relates to a cattle Nramp1 macrophage specificity expression vector as well as a construction method and application thereof. In the method, a plasmid pIRES2-EGFP (Internal Ribosome Entry Site-Enhanced Green Fluorescent Protein) serves as a skeleton carrier; a cattle Nramp1 open reading frame sequence is inserted between the multipe cloning site Sa1 I and BamH I; and the Nramp1 gene expression regulatory sequence is used for replacing a cytomegalovirus (CMV) on the skeleton carrier so as to realize the specificity expression of the Nramp1 gene in a phagocyte. The carrier can serve as a transgenic carrier to be applied in cultivating transgenic disease-resistant animals. The invention also points out that the similarity between the Qinchuan cattle Nramp1 open reading frame sequence and the corresponding sequence (of which the GenBank sequence number is U12862.1) in the GenBank is 99.88%, and two base differences exist.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to the construction of a eukaryotic expression vector, in particular to a phagocyte-specific expression vector of bovine natural immunity-related macrophage protein 1 (Nrampl), a construction method and application thereof. The carrier can be used as a transgenic carrier in the breeding of transgenic disease-resistant animals. Background technique [0002] To study the resistance of animal organisms to Mycobacterium bovis (M.bovis), Mycobacterium leprae (M.lepraemurium), Brucella (Brucella), Salmonella (S.typhimurium) and Leishmania spp (Leishmania spp) The infection mechanism of endoparasitic pathogenic microorganisms is the premise of seeking highly effective control drugs and vaccines and carrying out disease-resistant breeding of livestock. The body's defense against the infection of intracellular parasitic microorganisms mainly depends on cellular immunity, that is, macrophage...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
Inventor 王爱华李倩靳亚平张涌徐晓彬胡林勇
Owner NORTHWEST A & F UNIV
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