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Cloning and Expression of Cathelicidins Bac5 from Dairy Cattle and the Antimicrobial Activity Analysis

Author: YangLiHeng
Tutor: XueHuiWen；ZhangYong
School: Gansu Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Dairy cow Cathelicidins antimicrobial peptides Bac5 Clone The prokaryotic expression Antibacterial activity
CLC: S823
Type: Master's thesis
Year: 2011
Downloads: 31
Quote: 1
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Abstract


In recent years, due to the long-term indiscriminate use and abuse of anti-microbial agents, resistance increasingly serious problem, increase the difficulty of the prevention and control of animal diseases, antibiotic residues in animal products caused a serious impact on animal food safety. Therefore, the research and development of new, safe, effective and environmentally friendly antimicrobial imminent. The antimicrobial peptides widespread in plants and animals, and plays an important role in the body's innate immunity and acquired immunity attracting much attention, coupled with a strong antibacterial effect of drug-resistant strains. The cattle Bac5 a powerful extension of the spiral structure of cationic antimicrobial peptides antimicrobial peptides belonging Cathelicidins, the cattle body against foreign invading pathogens play an important role. The traditional method from bovine body tissues extracted Bac5, has complicated operation, high cost, low yield, not large-scale production and other shortcomings. Thus, by means of genetic engineering techniques to get Bac5 recombinant protein is expected to solve the problem, and has become a new development direction for the development of new environmentally friendly antimicrobial preparations. Included in this study is based on GeneBank bovine Bac5 gene sequence design and synthesis of a pair of specific primers, RT-PCR technology out the signal peptide Bac5 gene fragment was successfully amplified total RNA from Holstein young bulls bone marrow, EcoR Ⅰ and Xho Ⅰ digested prokaryotic expression vector orientation subcloned into pET28a constructed the pET28a-Bac5 recombinant plasmid, and the recombinant plasmid was transformed into E. coli DH5α. Positive clones were screened by PCR amplification, restriction enzyme digestion and sequencing, the recombinant expression in LB medium containing kanamycin (50μg/mL) plasmid pET28a-Bac5 was transformed into E.coli Rosetta, positive clones were screened PCR and sequencing again identified as positive for the bacterium IPTG induction, SDS-PAGE identification induced expression products, and take advantage of the E. coli O111 (CVCC1450) and Staphylococcus aureus (CVCC545) 2 standard strains and Escherichia coli, Staphylococcus aureus, Streptococcus mastitis milk common isolates preliminary identification of the antibacterial activity of recombinant protein expression Bac5. The results show that, from bovine bone marrow, the study successfully amplified target fragment and successfully build a the pET28a-Bac5 recombinant plasmid and sequencing results showed that the insertion of the target gene fragment size 414bp, encoding 137 amino acid residues, and GeneBank included The the bovine Bac5 gene sequence (L02650.1) homology is 100%, and the open reading frame is correct. Expressed by IPTG induction, the expression product was identified by SDS-PAGE, the molecular weight of approximately 19 KD, size consistent with the theoretical prediction. The bacteriostatic test results show that the common mastitis milk isolates to penicillin, streptomycin resistance, and the more sensitive the Bac5 recombinant protein; Bac5 recombinant protein of the two kinds of standard strains showed strong inhibitory bactericidal activity. The research laid the foundation for the clinical treatment of mastitis in dairy cows and the development of new environmentally friendly antimicrobial preparations and development.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Livestock > Cow
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