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HBD3 mammary gland specific expression vector and constructed recombinant cell

An expression vector and specific technology, applied in the field of genetic engineering, can solve problems such as negative effects, complicated results analysis, environmental safety problems of transgenic animals and plants, etc., and achieve the effect of avoiding potential safety hazards.

Inactive Publication Date: 2012-08-08
NORTHWEST A & F UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the selection of marker genes also brings some disadvantages: there are only a few effective marker genes widely used at present, and it is often necessary to perform multiple transformations on the same gene fragment during genetic engineering operations, and it is difficult to find suitable selection markers at this time. genes; in addition, the expression of selectable marker genes may have negative effects on the expression of other genes in transformed cells, leading to complicating the analysis of results; at the same time, the presence of selectable marker genes in transgenic crops increases the uncertainty of their distribution environment, This brings about environmental safety issues of transgenic animals and plants, which have a great restrictive effect on the environmental release of transgenic animals and plants

Method used

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  • HBD3 mammary gland specific expression vector and constructed recombinant cell
  • HBD3 mammary gland specific expression vector and constructed recombinant cell
  • HBD3 mammary gland specific expression vector and constructed recombinant cell

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Embodiment Construction

[0035] The HBD3 mammary gland-specific expression vector provided by the present invention first constructs the HBD3 mammary gland-specific expression vector pARNG-HBD3 with the attB sequence, and secondly makes the exogenous expression vector pARNG-HBD3 site Specifically integrated into the genome of bovine fetal fibroblasts, the expression of red fluorescent protein was observed under a fluorescent microscope, and positive cells were obtained through G418 screening, and PCR identification was performed to confirm that the target gene HBD3 was integrated into the genome of bovine fetal fibroblasts; then Positive cell clones were treated with Cre recombinase, and the antibiotic selection marker, red fluorescent protein gene and terminator between the two LoxPs in the same direction were removed, so that the Pcmv IE promoter and EGFP gene were spliced, and the green fluorescent protein was expressed. After flow cytometry The recombinant bovine fetal fibroblasts containing the HB...

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Abstract

The invention discloses a HBD3 mammary gland specific expression vector and a constructed recombinant cell. The vector comprises a HBD3 gene with a signal transduction peptide, and CSN5 and CSN3 are set upstream and downstream of the HBD3 gene respectively for serving as regulating and controlling elements. The host cell of the recombinant cell is a bovine fetal fibroblast cell, an exogenous expression vector pARNG-HBD3 is integrated to a pseudo attP locus under the action of phiC31 integrase, positive cloning is realized through medicament screening, and an antibiotic screening marker between two homodromous LoxP sequences on the vector is removed through Cre recombinase treatment. A HBD3-containing bovine fetal fibroblast cell from which an antibiotic screening marker is removed is taken as a nuclear donor cell of a transgenic cloning embryo through nucleus transplantation, gene cloning embryos are obtained through SCNT (Somatic Cell Nuclear Transfer), and a milk cow which is free from a HBD3 transgenic gene of the antibiotic screening marker can be produced by transferring these embryos into a receptor cow.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a site-specific integrated expression vector, in particular to a HBD3 mammary gland-specific expression vector and constructed recombinant cells. Background technique [0002] Mastitis is a very common disease in dairy cows. This inflammation is caused by a variety of pathogenic microorganisms, mainly Escherichia coli (E.coli) and Staphylococcus (S.aureus) (Bannerman DD, Paape MJ, Lee JW, Zhao X, Hope JC, Rainard P. Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clin Diagn Lab Immunol. 2004 May; 11(3): 463-72.). Mastitis has caused huge economic losses to the dairy industry. At present, the treatment of mastitis is still dominated by the use of antibiotics, but antibiotics are expensive; and long-term use of antibiotics will cause bacteria to develop drug resistance, so a new treatment for mastitis is sought...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/873
Inventor 余源张涌王勇胜刘军权富生
Owner NORTHWEST A & F UNIV
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