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Integration cattle NRAMPl macrophage specificity expression vector/cattle fibroblast and method thereof

A bovine fibroblast, fibroblast technology, applied in recombinant DNA technology, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as affecting the control effect, high virulence of strains, and unusable vaccines

Inactive Publication Date: 2012-07-11
NORTHWEST A & F UNIV
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Problems solved by technology

There are also a series of problems in the vaccination process (Zhao Fu; et al. Discussion on the Prevention and Control of Brucellosis and the Use of Vaccines. Veterinary Guide, 2010, 3: 24-27). Large, strong inoculation reaction, some vaccines are not very effective, some vaccines cannot be used for pregnant animals, and some vaccines cannot be used for adult animals, breeding males and diseased animals, etc. These factors limit the effectiveness of the vaccines. application, resulting in low inoculation density and affecting the control effect

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  • Integration cattle NRAMPl macrophage specificity expression vector/cattle fibroblast and method thereof
  • Integration cattle NRAMPl macrophage specificity expression vector/cattle fibroblast and method thereof
  • Integration cattle NRAMPl macrophage specificity expression vector/cattle fibroblast and method thereof

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Embodiment Construction

[0083] In the present invention, the NRAMP1 macrophage-specific expression vector pSP-NRAMP1-HA is first constructed, and the exogenous expression vector pSP-NRAMP1-HA is transfected into bovine fibroblasts by liposome method. Positive cells were obtained by G418 screening, and PCR identification confirmed that the target gene NRAMP1 was integrated into the genome of bovine fibroblasts. The fibroblasts transfected with NRAMP1 were transplanted into bovine enucleated oocytes as donor cells to obtain transgenic cloned embryos.

[0084] The specific reagents and materials involved are as follows:

[0085] G418, high glucose DMEM, Trizol, cell transfection reagent Lipofectamine 2000 and Opti-MEM R IReduced Serum Medium was purchased from Invitrogen Company of the United States; EDTA and Trypsin were purchased from Sigma Company of the United States; fetal bovine serum was purchased from Hyclone Company; cell culture plates and dishes were purchased from Corning Company; Triplemas...

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Abstract

The invention discloses an integration NRAMPl macrophage specificity expression vector / cattle fibroblast and a method thereof. An expression vector pSP-NRAMPl-HA containing NRAMPl macrophage specificity and a Qinchuan cattle fibroblast system which is based on the vector and contains NRAMPl are structured, and the method utilizing the cell system as a nucleus donor cell provides a solid foundation for solving transgenosis cattle problem suffering from Qinchuan cattle brucellosis. According to the invention, the exogenous expression vector pSP-NRAMPl-HA is conducted into the cattle fibroblast by a liposome transfection method, negative cells are obtained by G418 screening, and proved by PCR, the aim gene NRAMPl is integrated into the genome of the cattle fibroblast; and finally the cattle fibroblast cell integrated with the NRAMP1 gene is taken as the nucleus donor, a nucleus-removing oocyte is moved into the nucleus donor, thus obtaining a transgenosis clone body.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning animals, and relates to the construction of nuclear donor cells of transgenic cloned embryos, in particular to a bovine fibroblast integrating bovine NRAMP1 macrophage-specific expression vector. Background technique [0002] Brucellosis is a chronic infectious disease caused by Brucella abortus, which is zoonotic. Its clinical features are mainly miscarriage, long-term fever, orchitis, tenosynovitis and arthritis. Diffuse reticuloendothelial hyperplasia and granulomatous nodule formation. The tissues and organs involved in this disease are very extensive, and the monocyte-macrophage system, bone and joint system, and nervous system are the most common. Brucella is a small, short rod-shaped or club-shaped, non-spore-forming, Gram-negative bacillus. The pathogenic Brucellosis is divided into six types: bovine, sheep, pig, shilling, sheep and canine Brucella. Pathogenic bacteria are ma...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/06
Inventor 王华岩程祥邓捷马晓玲孟书燕来威锋
Owner NORTHWEST A & F UNIV
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