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Human lysozyme efficiently produced by utilizing mammary gland of domestic animal

A technology of human lysozyme and mammary gland, which is applied in the field of genetic engineering, can solve problems such as the lack of gene regulation methods, allergic reactions, and the difficulty in obtaining large quantities of natural human lysozyme.

Active Publication Date: 2009-12-16
SHANGHAI TRANSGENIC RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, lysozyme is restricted by three major factors when it is used clinically: 1) natural human lysozyme is difficult to obtain in large quantities
Currently human milk and placenta are the only sources to obtain natural human lysozyme (hLYZ), but these materials are rather limited, making the product prohibitively expensive
2) Lysozyme derived from other species can cause many side effects, such as egg white-derived can cause allergic reactions, and cattle-derived lysozyme has the threat of mad cow disease infection
3) Lysozyme derived from other animals is not suitable for human medicine, because the homology between them and human lysozyme is low, such as 56.8% from egg white, 76.4% from rats, and 82.3% from cows %, horse's is 50.8%
However, there is still no gene regulation method that can effectively make animals produce high levels of lysozyme

Method used

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  • Human lysozyme efficiently produced by utilizing mammary gland of domestic animal
  • Human lysozyme efficiently produced by utilizing mammary gland of domestic animal
  • Human lysozyme efficiently produced by utilizing mammary gland of domestic animal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1 Isolation of Saaneng Dairy Goat Fetal Fibroblast Cell Line

[0146] Male and female Saanen milk goats (Shanghai Transgenic Research Center Experimental Ranch) were bred 35 days later and the uterus of the ewes was surgically removed, and the uterus containing the fetus was put into sterilized normal saline. The goat fetuses were separated from the uterus, washed twice with PBS, sterilized with 70% ethanol, and washed twice with PBS. Use surgical scissors to peel off the fetal membranes in PBS, remove the head and liver, cut up the fetal tissue with tissue scissors, and spread it on a 100mm cell culture dish. Use GMEM culture medium containing 10% FBS, penicillin and non-essential amino acids at 37°C, 5% CO 2 , Cultivate under saturated humidity, observe the cell adhesion, replace the culture medium during the period, and remove the tissue blocks. As a result, when the primary cells were cultured to 80% confluence, observed under a microscope, the cells showe...

Embodiment 2

[0147] The construction of the mammary gland-specific expression vector of embodiment 2 human lysozyme gene

[0148] according to figure 1 Finally, the mammary gland-specific expression vector pbLGLyz of the human lysozyme gene was obtained, and the construction method was as follows: Design and synthesize the following primers according to the sequence in GENBANK accession number X14710:

[0149] 5'-cggccgggggtctgctcc-3' (SEQ ID NO: 1), and

[0150] 5'-ctcgagggctgcagctggggtcac-3' (SEQ ID NO: 2).

[0151]Using bovine genomic DNA as a template, the bovine β-lactoglobulin 5' flanking sequence (BLG-5' UTR) (-1215 ~ +45 bp), the PCR product was cloned into the pGEM-T-easy vector to obtain the plasmid pRBLGR. XhoI+SacII double-cut recombinant plasmid pTA-BGH obtained bovine auxin gene 3'UTR (PolyA sequence) (bGH-PA) with a size of 300bp, which was recovered and cloned into plasmid pRBLGR to form pRBLGpA. Finally, the human lysozyme gene (h-Lyz) was excised from pcDNA-LYZ with X...

Embodiment 3

[0153] Example 3 Co-transfected goat fetal fibroblasts and screening and identification of resistant clones

[0154] The expression framework of pbLGlyz was cut out with NotI enzyme, and the double marker selection vector pLNK( figure 2 ) expression frame was cut out, mixed according to the ratio (molar ratio) of pbLGlyz:pLNK=5:1, and electroporation method was used to co-transfect fetal fibroblasts, electric shock parameters: 0.4kV, 500uF. After the electric shock, let it stand for 10 minutes, wash out the cells with GEF complete culture medium, inoculate evenly in ten 100mm cell culture dishes, and place them at 37°C, 5% CO 2 Culture in an incubator; replace the selection medium (containing G418) 48 hours after transfection, and then replace the selection medium every two days until obvious cell clones are formed.

[0155] After the formation of cell clones, the clones were picked with a cloning ring, and passaged sequentially from the 96-well plate to the 48-well, 24-well...

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Abstract

The invention relates to a human lysozyme efficiently produced by utilizing the mammary gland of a domestic animal and discloses a fusion gene of mammary gland specific expression. The fusion gene sequentially contains the following elements from 5' to 3': a 5' flanking sequence of a beta-lactoglobulin gene of cattle, a human lysozyme gene and a 3' flanking sequence of a growth hormone gene of the cattle. The invention also discloses a method for preparing a transgenic animal and an efficient and safe transgenic animal preparation method for removing a marker gene.

Description

technical field [0001] The present invention belongs to the field of genetic engineering, and more specifically, the present invention relates to a mammary gland-specific expression fusion gene, a method for preparing transgenic livestock capable of efficiently expressing lysozyme in milk, and a highly efficient transgene for removing marker genes Livestock preparation methods. Background technique [0002] Natural lysozyme is a kind of polypeptide glycosidase widely existing in animal kingdom and plant kingdom. In 1922, Alexander Flemin discovered that a variety of mucus tissues and secretions, as well as egg whites, contained substances that could dissolve a variety of Gram-positive bacteria. He called this dissolution factor lysozyme. Flemin found that lysozyme exists in cartilage tissue and gastric homogenate, tears, saliva, sputum, nasal secretions, pathological urine, serum and lymphocytes, but it is not found in healthy urine, cerebrospinal fluid or sweat There is ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/56C12N5/10C12N9/36A01K67/027
Inventor 成国祥刘思国陈建泉俞慧清张爱民
Owner SHANGHAI TRANSGENIC RES CENT
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