Recombinant lysostaphin gene and expression vector and recombinant cell constructed by recombinant lysostaphin gene

Abstract

The invention discloses a recombinant lysostaphin gene and an expression vector and a recombinant cell constructed by the recombinant lysostaphin gene. The nucleotide sequence of the recombinant lysostaphin gene is shown as SEQ.ID.NO.1, a mammary gland-specific expression vector pBELS containing the recombinant lysostaphin gene and a green fluorescent protein (EGFP) gene is constructed, the mammary gland-specific expression vector pBELS is transfected to the ear fibroblast of a high producing dairy cow by using Fugen HD, the expression condition of labeled genes EGFP is observed by using a fluorescent microscope, the positive transgenic cells are obtained through G418 screening, the PCR (Polymerase Chain Reaction) and the Southern bloting identification are carried out, so that the condition that the recombinant lysostaphin gene is integrated into the genome of the ear fibroblast of the cow is confirmed. A positive transgenic cell in good growing state is taken as a donor cell, and a transgenic embryo is constructed and is implanted into a synchronized receptor cow uterus through embryo transplantation, so that cows with transferred lysostaphin genes are obtained.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning cows for studying anti-mastitis, and relates to a recombinant lysostaphin gene, an expression vector constructed therefor and a recombinant cell. Background technique [0002] Cow mastitis (mastitis), also known as cow mastitis, is mainly caused by the infection of pathogenic microorganisms. Inflammation caused losses reaching US$ 2 million (Sordillo LM, Streicher KL. Mammary gland immunity and mastitis susceptibility. J Mammary Gland Biol 2002; 7: 135-146). Cow mastitis is divided into clinical mastitis and subclinical mastitis. There are many types of microorganisms that cause mastitis in dairy cows. Staphylococcus aureus is one of the most important pathogens of mastitis in dairy cows. huge loss. [0003] Over the years, the treatment of cow mastitis has been treated with spectrum antibiotics. Although effective, the cure rate is less than 50% (Maidhof H, Reinicke B, Blumel P, Ber...

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Problems solved by technology

[0003] Over the years, the treatment of cow mastitis has been treated with spectrum antibiotics. Although effective, the cure rate is less than 50% (Maidhof H, Reinicke B, Blumel P, Berger-Bachi B, Labischinski H.femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. J Bacteriol 1991; 173: 3507-3513)

However, the mammary gland expression vector of lysostaphin directed by the BLG regulatory sequence and the somatic transgenic cell line of transfected animal cells, especially dairy cows, have not been reported; nor have the reports of transgenic cows with lysostaphin as the target gene

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