Virus and cells
The human type 5 adenovirus expression system (replication-defective) was purchased from TaKaRa (Dalian, China). Recombinant adenovirus (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% foetal 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.
Construction of recombinant adenoviruses (rAd-MSTN-SST)
The open reading frames of porcine MSTN (GenBank No. AY208121) were amplified by PCR using the primers (MSTN-SSTs and MSTN-SSTa, Table 1) with a plasmid harbouring artificially synthesized MSTN gene (General boil, Co.,Ltd, Anhui ) as templates. SST gene was fused into MSTN by a second round PCR reaction with the primers of MSTN-SSTs and MSTN-SSTa2. The PCR products were cloned into the transfer vector, pAd-shuttle-CMV. The recombinant adenoviral vectors were generated by homologous recombination of linearised transfer vectors with pAdEasy-1 in Escherichia coli BJ5183 strain and confirmed by restriction enzyme digestion (New England Biolabs). The recombinant adenovirus was generated by transfection of 1 μg plasmid (Pac I linearised) using 3 μL of Trans FastTM 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.
Table 1 Primers used for amplification MSTN-SST gene
Name
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Sequence(5'-3')
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RE Site
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Products size
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Primers properties
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MSTN-SSTs
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agatctatgcaaaaactg
|
Bgl II
|
|
Forward Primer
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MSTN-SSTa
|
tgagcacccacagcgatctac
|
|
1131bp
|
Reverse Primer
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MSTN-SSTa2
|
gatatcctaacaggatgtgaaagtcttccagaagaaattcttgcagccagctgagcacccacagcgatc
|
EcoR V
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1182bp
|
Reverse Primer
|
RE: restriction endonuclease
IFA
IFA was used to identify the expression of MSTN-SST in infected cells. Briefly, HEK293A cells were infected with rAd- MSTN-SST or wtAd (wild-type adenovirus) 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 mouse-anti-MSTN (Cloud-Clone Corp., MAB653Po21, 1:800 dilutions) and rabbit-anti-SST polyclonal antibody (Cloud-Clone Corp., PAA592Mu08M, 1:800 dilutions) mixture for 1 h at 37 ℃. Cells were stained with goat anti-mouse and goat anti-rabbit FITC-Conjugated Antibody (Abcam, ab6785, ab6717 1:800 dilutions) for 1 h at 37 °C. The cells were then washed with PBS and the expression of MSTN-SST was visualised under a fluorescence microscope (Olympus IX73).
WB
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% 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 antibodies for 2 h (Cloud-Clone Corp., MAB653Po21, 1:800 dilutions). 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 grey scan with Image J software and comparison with known concentration marker bands, the concentrations of the proteins were calculated according to the obtained grey values of the bands.
β-actin was used as a reference and the primary antibody against β-actin was purchased from Boster Co., Ltd. (Wuhan, BM0627).
Immunisation
Thirty 4-week-old male Chinese Kunming mice were purchased from Chengdu Dashuo Experimental Animal Co. Ltd. and randomly divided into three groups of ten. The mice had very similar weights. The first group received wtAd (at day 0) and served as a control group (Group A). The second group was given rAd-MSTN-SST at day 0 by intramuscular injection (Group B). The third group was given rAd-MSTN-SST at day 0 and boosted at day 14 (Group C). Mice were kept in cages with an automatic ventilation system. Five mice were kept in each cage and were allowed to feed freely.
The mice were weighed every week and blood was sampled on days 14 and 28. All the mice euthanised by CO2 inhalation. CO2 was infused in mice home cage at 10%, 30%, or 100% volume per minute displacement rates. When mice were in deep narcosis, they were sacrificed by cervical dislocation. To observe the muscles, the skins of the mice were removed and the muscle fibres (biceps femoris muscle) were observed by making tissue slices.
Histopathology tests
The collected biceps femoris muscles were fixed and embedded in paraffin wax and cut into 4–5 μm slices. For microstructural observation, the slices were stained with haematoxylin and eosin. The densities of muscle fibres in ten views were analysed using Image J software and the average densities of muscle fibre were used to express the increase in muscle quantity.
Enzyme-linked immunosorbent assay (ELISA)
ELISA plates were coated with the recombinant protein MSTN-SST (0.2 µg per well). The recombinant protein was expressed in E. coli. It was stored at −80 °C after purification until assayed. The sera of the mice collected at different times were detected with the ELISA plates as reported [12]. Commercial peroxidase-conjugated rabbit anti-mice IgG (Sigma, Shanghai, China) was used as the secondary antibody. After reaction, the absorbance values of the reaction system at 450 nm (OD450) were used to represent the antibody levels of the mice. Each sample was assayed in triplicate.
Statistical analysis
The bodyweights of mice are expressed as means ± standard deviation (SD) and evaluated with ANOVA. Differences between control and immunised groups were analysed by two-tailed independent Student’s t-tests. Differences were considered significant at P < 0.05.