2.1 Virus and cells
The human type 5 adenovirus expression system (replication-defective) was purchased from TaKaRa (Dalian, China). Recombinant adenoviruse (rAd) and wild type Adenovirus (wtAd) were grown and titered in HEK-293A cells.
All the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2.
2.2 Construction of recombinant adenoviruses (rAd-MSTN-SST)
The open reading frames of MSTN-SST were amplified by PCR using primers listed in Table 1. The PCR amplicons were cloned into a pAd-shuttle-CMV vector. The recombinant adenoviral vectors were generated by homologous recombination of linearized transfer vectors with the pAdEasy-1 in Escherichia coli BJ5183 and confirmed by restriction enzyme digestion (New England Biolabs). The recombinant adenoviruses were generated by transfection of 1 μg plasmids (PacI linearized) using 3 μL of Trans Fast TM Transfection Reagent (Promega, Madison, USA), When 90% of the cells showed cytopathic effect, adenoviruses were released by three cycles of rapid freezing and thawing, and stored at −80 ℃ after addition of 10% glycerol.
2.3 IFA
IFA was used to identify the expression of MSTN-SST. Briefly, HEK293 A cells were infected with rAd- MSTN-SST or wtAd at a multiplicity of infection (MOI) of 5. After 24 h incubation, the cells were washed, fixed with 4% paraformaldehyde (30 min at 25 °C) and incubated with the mouse-anti-MSTN-SST polyclonal antibodies (diluted 1:800) for 1 h at 37℃. Cells were stained with goat anti-mouse FITC-Conjugated Antibody (Abcam, ab6785, diluted 1:800) for 1 h at 37 °C. The cells were then washed with PBS, and the expression of MSTN-SST was visualized using a fluorescence microscope (OLYMPUS IX73).
2.3 Western blot
The 293A cells infected with rAd-MSTN-SST were collected at 18, 24 and 36 h post infection. The cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Pall Corporation). The 293A cells infected with wtAd were used as a negative control.
The membrane was incubated overnight in blocking solution (10% fat-free milk in PBS, PBS-M) at room temperature and incubated with anti MSTN-SST polyclonal mouse antiserum for 2 h. The membrane was subsequently reacted for 1 h with Goat anti-Mouse IgG conjugated with horseradish peroxidase (AS003) at a dilution of 1/2000 in PBS-M. Detection was performed using chemiluminescence lumi-nol reagents (Super Signal West PicoTrial Kit, Pierce). By gray scan with software Image J and compared with the known concentration marker bands, the concentration of the proteins were calculated according the obtained gray value of the bands.
β-actin was used as a reference and the primary antibody againstβ-actin was purchased from BOSTER Co., Ltd.. (Wuhan, BM0627).
2.6 Immunization
Thirty 4 week old male Chinese Kunming mice (male) were purchased from Chengdu Dashuo experimental animal co. LTD, and were randomly divided into 3 groups of 10. The mice were with very similar weight. The first group received wtAd (at day 0) and served as a control (Group A). The second group was given rAd-MSTN-SST at day 0 by intramuscular injection (Group B). And the third group were given rAd-MSTN-SST at day 0 and boosted at day 14 (Group C). Mice were kept in cages system with automatic ventilation system. Five mice were kept in one cage and they were free choice feeding.
The mice were weighted every week and blooding at day 14 and 28. The mice were gave euthanasia by using CO2 inhalation (CO2 were infused at 10%, 30%, or 100% volume per minute displacement rates) followed by cervical dislocation. To observed the muscles, the skin of the mice. The muscle fibers (biceps femoris muscle) were observed by making tissue slides.
2.7. Histopathology test
The collected biceps femoris muscle were fixed and embedded in paraffin wax and cut into 4 to 5 μm slices. For micro structure observation, the slices were stained with haematoxylin and eosin. The densities of muscle fibers in one view were analyzed using software Image J.
2.9. Enzyme-linked immunosorbent assay (ELISA)
ELISA plates were coated with the recombinant protein, MSTN-SST (0.2 µg / each well). The recombinant protein was expressed in Escherichia coli, and after purification, it was stored at minus 80°C until assayed. The sera of the mice collected at different time were detected with the ELISA plates. Commercial peroxidase-conjugated rabbit anti-mice IgG (Sigma, Shanghai, China)) was used as the secondary antibody. After reaction, The absorbance value at 450 nm (OD450) were used to present the antibody levels of the mice. Each sample was assayed in triplicate.
2.10. Statistical analysis
The body weight of mice was expressed as mean ± standard deviation (SD) and evaluated with ANOVA. The body weight of mice was, and the differences between control groups and immunized groups were analyzed by a two-tailed independent Student’s t-test. A difference was considered to be significant if P < 0.05 was obtained.