Expression vector construction
The pGEX-5X-1 vector was purchased in Amersham Pharmacia Biotech (Piscataway, New Jersey, USA) and was then amplified in LB culture medium (tryptone 1%, yeast extract 0.5%, NaCl 1%, pH: 7.0). HBD3 sequence was obtained by mature protein sequence, and optimal sequence was synthesized in Invitrogen Company (Invitrogen, Carlsbad, CA). The optimal designed sequences are as follows: (GTGATCATTAACACTCTGCAAAAATATTACTGCCGCGTGCGTGGTGGCCGTTGTGCGGTTCTGTCCTGTCTGCCGAAAGAAGAGCAGATCGGCAAATGCTCTACCCGCGGTCGTAAATGCTGCCGTCGTAAAAAGTAATGATGAGAATTC). The vector and optimal sequence were double digested by BamHI, EcoRI and BclI, EcoRI separately and were connected by T4 ligase enzyme. Recombinant of pGEX-5X-HBD3 plasmid was identified by digestion and sequencing.
Determination of optimal induced conditions
Optimal-induced condition test was operated to obtain more soluble HBD3 protein. In the previous study, the optimal-induced temperature and IPTG concentration were tested. Hence, we selected 28 °C and 1 mM IPTG as optimized conditions, and the optimal inducement time was evaluated. Bacteria were collected at 3, 5, 7, and 9 h, and then the bacteria were cracked by lysozyme. Supernatant and sediment were separately collected after being centrifuged on 4000 rpm for 5 min. Optimal induce time was analyzed by SDS-PAGE.
Purification of GST-HBD3 fusion protein
The E. coli bacteria that containing pEGX-5X-HBD3 plasmid was amplified in LB medium (0.5% yeast extract, 1% tryptone, 1% NaCl) containing 50 μg ampicillin/ml. IPTG was then added into the medium when its OD (600) reached 0.4. The bacteria were harvested and collected by centrifuging at 4000 rpm for 20 min. The purified GST-HBD3 recombinant protein was obtained as previously reported (Huang et al. 2007).
Identification and harvest of rHBD3 protein
Purified HBD3 recombinant protein was harvested by cleavage of GST-HBD3 protein using Factor Xa (NEB) at room temperature. The enzyme and Xa buffer (20 mM Tris–HCl: pH 8.0 with 100 mM NaCl and 2 mM CaCl2) were incorporated into the GST-HBD3 protein at 23 °C for 6 h, and the total digested protein was dialyzed by binding buffer (50 mM NaH2PO4–Na2HPO4, 1 M NaCl, pH 7.4). The purified protein was obtained after flowing through Sepharose® Fast Flow system. The protein was checked by Tris-tricine-SDS-PAGE and was confirmed by western blot analysis. After being transferred on a PVDF membrane, the protein was confirmed after incubation with HBD3 antibody (Sigma, St. Louis, MO) at 4 °C overnight and with goat anti rabbit IgG (Sigma, St. Louis, MO) for 2 h.
Antimicrobial activity of purified rHBD3 on BL21 (DE3) E. coli and Staphylococcus
Antimicrobial activity tests of HBD3 protein were evaluated for its biological function by a bacteria growth curve test for BL21 host bacteria and Staphylococcus aureus (ATCC25923). Exponentially growing bacteria were re-suspended in 10 mM sodium phosphate buffer (pH 7.4) to reach a density of 5 × 107 CFU/ml. Ten microliters of each bacterial suspension was exposed for 1.5 h under the appropriate culture condition to different treatments in 100 μl of 10 mM sodium phosphate buffer (pH 7.4). The number of bacteria as directed by the optical density (OD 600) was measured every 30 min. The inhibition zone tests were performed to identify its inhibitory concentration.
Determination of minimal inhibition concentration (MIC) on M. bovis
Mycobacteria bovis virulent strain C68014 were purchased from the China Institute of Veterinary Drugs Control (Beijing, China) and cultured on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI) for 20 days. The colonies were then transferred to Middlebrook 7H9 modified medium (Difco Laboratories, Detroit, MI, USA) for 20 days. Determination of MIC was performed in M. bovis growth curve. Serial concentrated peptide dilutions in Middle Brook 7H9 broth were prepared. Subsequently, 50 μl of this suspension was mixed with 50 μl of the peptide dilution in each well. After incubation for 12, 24, 48, 60, 72, 84, and 96 h at 37 °C, the bacterial number directed by OD600 data was measured.
In vitro infection of A549 cells
Human type II alveolar pneumocytes A549 (CCL185) and RAW 264.7 cells were separately cultured in 75 cm2 culture flasks (CAS, Shanghai, China) with antibiotic-free Dulbecco’s modified Eagle’s medium (HyClone laboratories, Logan, Utah) supplemented with 10% fetal calf serum (Gibco BRL, Grand Island, NY). A549 and RAW 264.7 cells were pre-incubated in the medium for 24 h prior to M. bovis infection. The cells were infected with M. bovis at MOI 10:1 for 24 h. M. bovis was then detected by Auramine O (Sigma) methods according to the manufacturer’s instructions.
Cell apoptosis detection
Mycobacteria bovis (MOI: 10:1) and HBD3 protein were separately co-incubated with A549 and RAW 264.7 cells for 24 h. Cell apoptosis was then evaluated by DeadEnd TM Fluorometric Tunel System (Promega Corporation, Madison, WI, USA) and cell nucleus dying by PI according to the instruction book (Sigma Co, St. Louis, MO). Cell apoptosis ratio was then detected by flow cytometry.
Statistical analysis
Inhibited concentration, FCM and CFU test results were analyzed using SPASS software (SPSS, Chicago, IL, USA). The changes are presented as mean ± SEM and were compared using one-way ANOVA followed by Newman–Keuls test. P values < 0.05 were considered as statistically significant.