Expression Of Viral-mediate System For Co-regulation GH In Mouse Skeletal Muscle And Its Effect On Animal Growth-Enhangcing

Somatostatin (SST) and growth hormone-releasing hormone (GHRH) are synthesized and secreted by the hypothalamus, which can control the synthesis and secretion of the growth hormone (GH) from the hypophysis as well as regulate the GH concentrations in animals and humans. GHRH controls pulsatile secretion of GH, and SST controls basal secretion of GH. The gene expression of GHRH is inhibited by antagonistic action from SST. A lot of researches confirm that GHRH peptide and antibodies against SST expressed in vivo by gene transfection technique can improve animal productivity. In order to cut down antagonistic action from SST and improve growth hormone level preferably, we project that expressing GHRH peptide and immune neutralizating somatostain in vivo simultaneously. It is able to implement the two-ways regulation for GH and may be a potential way to increase animal productivity.Because skeletal muscle has good capacity for protein synthesis, is easily accessible for intramuscular injection, and has the ability to take up plasmid after intramuscular administration, it is an attractive tissue for somatic gene delivery. The cytomegalovirus (CMV) enhancer/promoter (E/P) is generally considered to be the most powerful promoter for gene therapy in muscle, but interestingly the presence of multiple CMV enhancers does not improve gene expression further. Many efforts have been made, with some progress, to improve the level of exogenous gene expression in muscle through the development of novel gene expression systems. For example, a synthetic muscle- specific promoter shows more persistent expression than the CMV promoter. In our reaserch, we tested a simple idea of whether inclusion of additional promoter, muscle- specific promoter-SP, behind of CMV promoter may enhance the expression efficacy of an exogenous gene in mouse skeletal muscle. Compared with single promter vectors, corresponding two-promoter vectors yielded Lac Z as reporter gene in vitro and vivo. To elevate the SP promoter tissue specificity, we transient transfections in several nonmuscle cell lines. In the 293T line (human transformed embryonic kidney), BHK line (baby hamster kidney) and HeLa cells (human cervix epitheloid carcinoma), specificβ-galactosidase (β-gal) activity of constructs was relatively low compared with the prevalently expressed CMV promoter.But in C2C12 line (Mouse myoblast cell line),β-gal activity of the two pomoters construction was significantly high than single promoter constructions, respectively. In vivo expression of two promoters was compared with that of the promoters CMV and SP promoter, after direct intramuscular injection in adult immunocompetent mice. At 2 and 4 weeks postinjection, the CMV/SP–driven construct had an activity 2.23 to 2.60 fold higher than that of the CMV promoter and 1.98 to 2.23 fold greater then that of the SP promoter. These results indicate that two promoters construction can enhance exogenous gene in mouse skeletal muscle efficiently.Due to its non-pathogenicity, the ability to infect various types of cells, recombinant adeno-associated virus type (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have constructed AAV-GHRH, AAV-HBsAg/SST and AAV-GHRH-HBsAg/SST recombinate AAV particle and then studied its effction on regulating animal growth. SDS-PAGE was used to monitor the purity of AAV-GHRH, AAV-HBsAg/SST and AAV-GHRH-HBsAg/SST respectively. The blot hybridization was used to determine the physical titers of AAV-GHRH, AAV-HBsAg/SST and AAV-GHRH-HBsAg/SST. The results of SDS-PAGE and HPLC showed that the parities of the final rAAV 2 products were higher than 99%. The physical titers of three AAV vectors were 1×10¹² virus genomes/mL (vg/mL), respectively.The transduction efficiencies of AAV-GHRH-HBsAg/SST were studied via intramuscular injection. We monitor the weigt gain per day of mice, expressed level of GHRH and HBsAg/SST in vivo yield semi-quantitative RT-PCR. Meanwhile, we detected the presence of vector and presence of expression in vivo. The results that increase in weight gain/day by AAV-GHRH (10⁸vg/mouse) and AAV-HBsAg/SST (1010vg/mouse) was observed, the vector and expression were merely showed in injection point.There was no sigfinant increase were observed in group of AAV-GHRH-HBsAg/SST. The reason is that in insert sequerence was too large to exceed the vector capability.Then we injected AAV-GHRH (10⁸vg/mouse) and AAV-HBsAg/SST (10¹⁰vg/mouse) simultaneously in same animal. The result of mice weight gain per day confirmed that promoted growth in animal due to GHRH and HBsAg/SST gene transfer mediated by AAV. Immunohistochemistry from muscle injected site were detected. The result confirmed that interest protein corresponding were expressed in the mice muscle. After 30 days, the weight gain per day in those groups treated with two vectors significantly 87.98%(P<0.01) higher than the group injected with PBS and two single gene AAV vectors. In all 40 days, Concentrations of serum IGF-I in the group treated with two vectors were higher than three other groups (116.24%, 105.50%, 104.08%, P<0.01). The result of correlation analysis showed that after injected the co-regulate construction, the weight gain was significantly related to the IGF-I positively (P<0.05), the corelative coefficient was 0.881. The result of ELISA and RIA detection for serological specificity HBsAb and anti-SST were positive after injected 2 week. 30 day post-injection, the level of serum SST were significantly lower than other groups (P<0.05). The results indicated that the combination of AAV-mediated GHRH supplementation and positive immunization against SST led to more robust weight gain per day in mice.Lentiviral vectors are attractive tools for human gene transfer. We constructed the recombinant lentiviral vectors of GHRH gene.The recombinant GHRH vector was regulated by CMV promoter and SP promoter. High titer of recombinant lentivirus was prepared from 293T cells by Lipofectin-mediated transient cotransfection of four plasmids, and purified by ultracentrifugation .The viral titer was measured by fluorescent microscopy. GFP expression was detected in 293T, BHK, and C2C12 cell lines, which were infected with recombinant LV-SP-GHRH-GFP. To evaluate effect on enhance animal growth by LV- SP-GHRH-GFP, mice were administered of viral vector. The results showed that 28 days post-injection weigt gain per day of mice injected LV-SP-GHRH-GFP (10⁷ifu/mouse) were significantly higher than the group injected PBS, the group injected LV-GFP and even higher than Group AAV-GHRH. Thus, lentiviral mediated gene transfer provides a powerful tool for the production of regulate animal growth.In summary, the results showed that the introduction of AAV mediated GHRH and HBsAg/SST fusion genes into mice muscles not only resulted in the expression of GHRH but also simultaneously induced antibodies to neutralize endogenous SST, and thus it provided the highest serum IGF-I level and the greatest weight gain/day. Furthermore, Lentivirus mediate GHRH could be a novel way to enhance animal growth efficiency. It is a strong indication that viral vector mediated GH co-regulation constructions may be potential drugs that function as growth stimulants in animals.