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Bovine fetal fibroblast containing an HLZ mammary gland specific expression vector

A technology of fibroblasts and expression vectors, applied to cells modified by introducing foreign genetic materials, using vectors to introduce foreign genetic materials, and methods based on microorganisms, etc., can solve the problems of low lysozyme content and no significant effect of bacteria

Inactive Publication Date: 2010-11-24
NORTHWEST A & F UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The content of lysozyme in the human body is very high, reaching 400μg / ml, which can effectively inhibit the growth of bacteria, but in ruminants, the content of lysozyme is very low, and has no significant effect on bacteria

Method used

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  • Bovine fetal fibroblast containing an HLZ mammary gland specific expression vector
  • Bovine fetal fibroblast containing an HLZ mammary gland specific expression vector
  • Bovine fetal fibroblast containing an HLZ mammary gland specific expression vector

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Embodiment Construction

[0032] The present invention constructs the expression vector pCSN2-HLZ-Neo-TK through the method of gene targeting. In the expression vector, the 5' end of the target gene HLZ gene is inserted into the CSN5 sequence, and the 3' end is inserted into the CSN3 sequence as a promoter, and the CSN5 sequence and the CSN3 sequence are They are respectively the 5' and 3' regulatory regions of the goat-casein gene CSN2 promoter. Since β-casein is the main protein in milk of cows, the bovine β-casein gene has a strong expression activity and is highly conserved. The homology of the β-casein gene sequences of cattle and sheep is 99%. , so the exogenous gene can be integrated into the genome of bovine lactating cells through homologous recombination, and then the specific expression of HLZ gene can be induced under the stimulation of prolactin and hydrocortisone. Therefore, the CSN5 sequence and the CSN3 sequence have regulatory effects on their downstream target genes, so that they can ...

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Abstract

The invention discloses a bovine fetal fibroblast containing an HLZ mammary gland specific expression vector. A CSN5 sequence and a CSN3 sequence serving as regulating elements are inserted at the 5' end and the 3' end of the HLZ gene by constructing the HLZ mammary gland specific expression vector respectively; and the CSN5 sequence and the CSN3 sequence have regulating effects on downstream target genes thereof so as to realize specific efficient expression in bovine mammary gland tissues. The expression vector pCSN2-HLZ-Neo-TK is transfected to the bovine fetal fibroblast by using an electroporation method, then the bovine fetal fibroblast is subjected to G418 and GVC double-medicament screening to obtain a positive cell, the positive cell serving as a nuclear donor is transferred to a bovine enucleated oocyte to obtain a transgenic clone embryo, the accuracy of genetic integration of the transgenic clone embryo is identified by PCR, and the transgenic clone embryo is transferred into a bovine womb of a receptor so that a transgenic dairy cow of which mammary tissues secrete lysozyme is expected to be obtained.

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 fetal fibroblast containing HLZ (lysozyme) mammary gland-specific expression vector. Background technique [0002] Mastitis is a transmissible bacterial inflammation mainly caused by staphylococcus, streptococcus and coli. Bacterial mastitis has brought huge economic losses to the production of animal husbandry because pathogenic bacteria widely exist in livestock houses and have a long incubation period and are difficult to eradicate. At present, the main preventive and treatment measures for mastitis in dairy cows are the use of cephalosporins or lactam antibiotics, but the long-term use of antibiotics will cause drug resistance of pathogenic bacteria and the problem of drug residues in milk, so it is necessary to seek a new Methods of treating mastitis. [0003] B...

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/877C12R1/91
Inventor 武志强张涌孙达权
Owner NORTHWEST A & F UNIV