Adenovirus Virus Expressing Myostatin- Somatostatin Fusing Gene Promote the Growth Rate of the Mice CURRENT

Background Myostatin (MSTN) and somatostatin (SST) are all involved in regulation the growth of the animals. Animals immunized with these proteins induce the related immune response, which could neutralize the normal inhibitory effects of these two proteins. Here, a recombinant adenovirus expressing MSTN-SST was constructed and used to deliver these two antigen to mice, so as to check the immunize efficacy of these proteins delivered in this way. Results The adenovirus expression fusing protein MSTN-SST was successfully rescued on HEK293A cell as adenovirus related cell pathological change were observed after transfection with the adenovirus genome harboring MSTN-SST gene. The expression of MSTN-SST were confirmed using western blotting and indirect immunofluorescence. Immunization with the recombinant adenovirus once or twice were successfully induce immune response against MSTN-SST and resulted in an improved growth rate and muscle mass. In addition, a booster immunization leaded a better results shown as higher antibody response and mice growth rate. Conclusions Accordingly, adenovirus can be used as a vector for deliver myostatin and somatostatin gene to increase animal growth rate and muscle mass .


1.background
Myostatin (MSTN), a member of transforming growth factor (TGF)-ß superfamily, is also known as growth and differentiation factor 8 (GDF8) [1]. MSTN was found to be capable of modulating the body weight and muscle composition in laboratory and farm animals [2]. Oral feeding recombinant yeast Saccharomyces cerevisiae expressing mammalian MSTN from a plasmid or chromosomal integration gene elicited antigen specific immune responses, and resulted in increased body weight and muscle composition in mice [3,4]. Downregulation of MSTN expression by siRNA or gene knock out can also increase muscle mass [2,5]. Accordingly, MSTN is an ideal target for regulation animal meat product or human muscle wastage. Somatostatin (SST) is known to inhibit the release of growth hormone (GH) from the anterior pituitary [6]. Reduction of the concentration of SST in the blood results acceleration of the growth of the animals. Immunization of animals to SST and a result induction of the related antibodies (the anabolic factors) is a means of removing SST's normal inhibitory effects. However, SST is a short peptide 3 hormone with only 14 amino acids and its half-life in the blood stream is only several minutes. So SST conjugates with various proteins are used for immunization [7]. Here, MSTN were fused to SST, so as to induce the related immune response and acceleration of the growth of animal and enhancement meat product.
Adenovirus vectors are the most commonly employed viral vector for gene therapy and deliver of vaccine antigens. They have been used for delivery of rabbit hemorrhagic disease virus antigen VP60 [8] and many other virus antigens like, antigens of porcine reproductive and respiratory syndrome virus [9] or foot-and-mouth disease virus [10]. And the immunization with recombinant adenovirus induced robust immune response and also protection against infection. Here, we used adenovirus to deliver MSTN and SST, and which may induce a strong immune response to regulate the growth rate of the immunized animals.

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% CO 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 4 stored at −80 ℃ after addition of 10% glycerol.

Western blot
The β-actin was used as a reference and the primary antibody againstβ-actin was purchased from BOSTER Co., Ltd.. (Wuhan, BM0627).

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 5 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.

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.

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 (OD 450 ) were used to present the antibody levels of the mice. Each sample was assayed in triplicate.

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.

Humoral immune responses against MST-SST following vaccination
The sera were collected at 7, 14, 21 and 28 days post immunsation and were detected by ELISA with the recombinant proteins (E.coli expressed, 0.2 µg in one well) as reported [11].
The antibody against MSTN-SST was detected as early as 7 days post inoculation. And the antibody titer reached a peak at day 21 with only one time immunization as that shown in group B (Fig 2).
Antibody titer was still increasing even at end of the experiments in group C, which were immunized twice. The immunized groups gained significant high level of antibody titer since 7 days post inoculation and till to the end of the experiment (P≤0.05). The immunized two groups showed significant difference since day 28 (P≤0.05) (Fig 2).

Body weight gain
The experiment lasted a total of 4 weeks before the mice were sacrificed. The body weights of each animal were recorded each week. When the experiment started all mice were about the same size with little difference. At the end of experiment, however, they weighted from 34 to 42.3 g and the average sizes varied among groups. In Group A, the average increase of body weight was 14.18± 0.36 g, whereas it was 16.70 ± 0.252 g in Group B, suggesting that vaccination against MSTN-SST modulated the body weight of animals. Interestingly, the increases of body weight in Groups C were significantly higher than that of Group B, suggesting that boosting immunization was effective and further confirm the role of MSTN-SST on regulation growth rate of the mice (Fig 3).

Muscle morphology observation
To show the effect of the immunization on the growth of mouse muscle, post-mortem examinations were performed on these animals. As show in Fig 4,

Histology examination
Histology examination was used to show the micro-level differences among the different groups. As show in Fig 6, the muscle fibers of BF from each group were observed under microscope. The thicken muscle fibers in group B, C were observed when compared the muscle collected from group A. In addition, the densities of muscle fibers were significantly increased as the numbers of the fibers were significantly increased in one view. According to statistical results of 10 views, the proportions of muscle fibers were increased from 63.43±3.28 to 72.18±4.28 and 83.72±1.67, respectively. And the differences were significant among each group.

Discussion
Adenovirus vectors are the most commonly employed viral vector for gene therapy and delivery of vaccine antigens. Adenovirus vectors used as vaccines are mostly replication-defective with certain 8 essential viral genes deleted and replaced by a foreign gene expression cassette [12,13]. Here, the MSTN-SST gene fusion was inserted into the Ad5 genome, which was used to express the MSTN-SST protein. Immunization with recombinant adenovirus induced an antibody response against the recombinant MSTN-SST protein. This resulted in accelerated gain of body weight and muscle mass in mice. In previous research, MSTN were delivered though heat-killed whole recombinant yeast Saccharomyces cerevisiae expressing mammalian MSTN from a plasmid, and which elicited antigen specific cell and humor response in mice [4]. The immunization increased body weight and muscle composition in mice. Soon after, another reports demonstrated that heat-inactivated MSTNrecombinant yeast could promote the growth of the rabbits and significantly increase the development of muscle [14]. And then, saccharomyces cerevisiae harboring MSTN in the genome were constructed and used to deliver MSTN gene to mice by oral immunization [3]. Similar results happened to the immunized mice were observed. In addition, monoclonal anti-MSTN antibody injected into the yolk significantly increased body weight (4.2%) and muscle mass (5.5%) [15]. Though the oral immunization with Saccharomyces cerevisiae or directly given monoclonal antibody could induce immune response against MSTN and acceleration of growth of the animal, the immune operation is not easy to carry out in field. Immunization by muscle injection is most used in clinical production, because it is easy to standardize. Here, adenovirus expression MSTN-SST was given by intramuscular injection and all the immunized mice gained similar antibody level. Animals immunized with SST had an increased average daily weight gains of 10-20%, and appetite reduced by 9% and an 11% increase in the efficiency of food utilization [7]. Wherein improved absorption of food components and a slower passage of food through the gastrointestinal tract with sluggish peristalsis is observed. Animals immunized with SST, and also their offspring, have correct proportions, and the distribution of the weight of the animals between the muscles, bones and fat is the same as in the control [1,7]. Another research found that immunization of pregnant goats resulted in an increased in the weight of newborn by 10% and an increase in milk yield [7]. Here, according to our results, immunization with MSTN-SST improved daily weight gains of the mice and also the gross weight of the mice. However, we did not measure the food consumption and the mice 9 were allowed to feed freely.

Conclusion
We constructed an adenovirus which expressed fusing proteins, MSTN-SST. Mice immunized with this adenovirus had enhanced daily weight gain and muscle mass. This recombinant adenovirus can be used to increase animal meat production, and may also can be used for treating muscle atrophy.

Authors' contributions
GX and NZ conducted the experiment. XC and DY did the data analysis and XW prepared the manuscript.    Display muscle shape of the mice by removing the skin. Random selected four mice from each group were used to show the muscle shape. The skin of the mice were removed after given euthanasia. The date collected from group A, B, and C were shown as A, B and C in figure. The differences of the muscle between the groups can be visually seen.

Figure 5
The size of Longissimus dorsi (LD) and Biceps femoris (BF) of mice from different group. LD and BF from each mice were collected and weighted. Immunization significant increases the size of these two type of muscle. The date collected from group A, B, and C were shown as A, B and C in figure. Data are shown as Mean±SD. Significant difference (p≤0.05) were marked with * Figure 6 Display the change of muscle fibers shape by histological examination. BF collected from each group were fixed and transected to make slices. The slice from group A, B, and C were shown as A, B and C in figure. The muscle fibers were observed under microscope. The increased muscle fiber densities were observed in immunized groups.

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