| Bovine mastitis is one of the most three common diseases in the dairy industry. Moreover, coliform mastitis is the most prevalent form of clinical mastitis, with infection caused by Escherichia coli being the most frequent pathogen. Approximately 80% of all intramammary infections by coliform bacteria will result in clinical mastitis, and about 10% of mastitis cases will become peracute with the sudden onset of severe clinical symptoms of endotoxin shock. The cellular receptor for lipopolysaccharide (LPS) of Gram-negative bacteria is CD14 which is typed into membrane-CD14(mCD14) expressing on monocytes/macrophages and neutrophils and souble CD14(sCD14) present in serum, urine, and milk. The cellular response to LPS is modulated by interactions among LPS, CD14 and LPS-binding protein (LBP) which is an acute phase protein. Accumulated evidence shows that sCD14 competes with mCD14 for LPS and plays a pivotal role in regulating bacterial infection and septic shock caused by Gram-negative bacteria. There were several reports involved in recombinant bovine sCD14(rbsCD14) being applied for the control of bovine mastitis at abroad, however, no information in detail in China.In this study, leukocytes from bovine infected coliform mastitis were isolated, total RNA was extracted and synthesized for cDNA. According to the sequence of bovine CD14, the primers were designed for amplifying bovine sCD14. The products of PCR were cloned into pMD18-T easy vector. Positive recombinant clones with the size of 1026bp insert of sCD14 gene were identified and confirmed by restriction enzyme digestion and sequencing. The target gene was inserted into baculovirus transfer vector pFastBacHTa. Then positive recombinant transfer vector pFastBac-sCD14 with sCD14 gene was further confirmed and transformed into DH10Bac E.coli cells carrying a baculovirus shuttle vector (Bacmid) and a helper plasmid. sCD14 gene fragment was integrated into Bacmid by site-specific transposition and the recombinant Bacmid was named pBacmid-sCD14. The recombinant baculovirus, designated rBac-bsCD14, was obtained from Sf9 insect cells transfected with pBacmid-sCD14 mediated by lipofectin. Entry of pBacmid-sCD14 DNA into cells was identified by PCR, and the recombinant protein expressing in Sf9 cells was identified by Western blot. Subsequently the resulting purified recombinant bovine sCD14(rbsCD14), approximately 1.1mg, was performed by chromatography of metal affinity resin, dialysis and PEG precipitation.Furthermore, the biological function of the recombinant protein was evaluated in mastitis animal model. To investigate the protective effect of rbsCD14 against LPS, the ten eight-week old BALB/c female mice were randomly assigned to two groups, injected intraperitoneally with either LPS (8μg/g of body weight, n =3) plus PBS or LPS plus rbsCD14 (6μg/g of body weight, n=3). During the period of 24h to 48h post challenge, the death account was 5 and 3, respectively. To investigate the protective effect of rbsCD14 on experimentally induced mastitis in mice, two abdominal contralateral mammary glands of four multi-baby-mother BALB/c mice were injected intramamary with 25 colony-forming units (CFU) of Escherichia coli strain P4. One gland received rbsCD14 (6μg) and the other for PBS simultaneously. At 24h after challenge, glands that received rbsCD14 had less swelling and hemorrhaging, significantly lower bacterial number by counting, compared to the control group without rbsCD14 treatment. Results show that rbsCD14 presumably binds or neutralizes LPS in evaluation of cell bioactivity and reduces mortality in mice from endotoxin shock and severity of intramammary infection by E. coli. They also indicated that rbsCD14 could be a potential therapeutic agent to minimize the impact of bovine mastitis caused by Gram-negative bacteria.
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