【作者】 任晓慧；

【导师】 张永亮；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 本研究采用塞姆利基森林病毒(SFV)RNA复制子和微球(MP)介导基因转移技术,以期提高目的基因的表达水平。首先构建了GHRH和HBsAg-SS单基因以及GHRH-HBsAg-SS双基因RNA复制子真核表达载体,在细胞和小鼠肌肉中成功地表达了目的蛋白,且表达的HBsAg-SS具有反应原性。制备的MP-DNA复合物提高了目的基因的表达以及基因免疫水平;也证实了复制子载体对基因表达具有明显的促进作用,表达水平比普通载体高3倍,诱发抗体水平的升高也显著优于普通载体。微球介导的GHRH和HBsAg-SS基因直接转移,对小鼠和育肥猪均表现出明显的促生长作用。初步探讨了非遗传性获得的促生长机制,证实利用这项技术在大型哺乳动物产生两代间的,非遗传性生物效应的可能性。给妊娠母鼠和母猪进行GHRH和HBsAg-SS基因直接转移,后代表现出明显的促生长作用。同时也证实了GHRH与HBsAg-SS双基因共表达对动物的促生长具有协同作用。本研究为寻求高效的基因表达系统,实现对GH的双向调节,开发新一代提高动物生产性能的长效促生长制剂打下了基础。更多还原

【Abstract】 Growth hormone(GH) administration has widespread use in livestock production to improve the efficiency of dietary nitrogen utilization while promoting greater protein accretion and milk production. Growth hormone (GH) is released in a regulated, distinctively in an impulse type. This phenomenon is under the control of the hypothalamic hormones, the functional interplay of two hypothalamic hypophysiotropic hormones, GH-releasing hormone (GHRH) and somatostatin (SS), exerting stimulatory and inhibitory influences, respectively. GHRH controls pulsatile secretion of GH, and SS controls basal secretion of GH. The gene expression of GHRH is inhibited by antagonistic action from SS. A lot of research confirm that GHRH peptide and antibodies against SS expressed in vivo by gene transfection technique can improve animal productivity. In order to cut down antagonistic action from SS 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.Gene transfer technique mediated by microsperes and RNA replion were introducted to our study. The eukaryon expression vectors based RNA Replicon, pCMV-Rep-GHRH, pCMV-Rep-HBsAg-SS and pCMV-Rep-GHRH-HBsAg-SS were constructed respectively in our investigation. The interest protein that expressed in eukaryon cells and skeletal muscle of mice successfully have biological activity by detecting. The result confirmed that transgene expression from GHRH and HBsAg-SS mediated by PLGA microspheres in skeletal muscle have growth-enchancing effect significantly for mice and pigs.We have demonstrated that pregnant rat dams treated with this system give birth to pups that have significantly increased numbers of somatotrophs and lactotrophs, as well as long-term increase in body weight and concentrations of serum IGF-I. We also have demonstrated that similar effects have been obtained in the porcine model despite a longer gestation time and different placental structure compared with the rodent model. The average birth weights, concentrations of serum IGF-I and GH, growth rate of piglets which were born to gilts that had received GHRH and HBsAg-SS plasmid were significantly heavier than control groups. The results aslo demonstrated that the enhanced gene expression, genetic immunizationand and ability protecting plasmid DNA from degraded by nuclease due to microspheres adsorption and RNA replicon may significantly promote gene expression. We can also draw another conclusion that a synergistic action is displayed between GHRH and SS from co-expression vector pCMV-Rep-GHRH-HBsAg-SS in growth-enchancing and improve immunity in mice and first-generation progeny pigs.The vectors construction of pCMV-Rep-GHRH, pCMV-Rep-HBsAg-SS and pCMV-Rep-GHRH-HBsAg-SS. GHRH or HBsAg-SS gene fragment was obtained by PCR amplification, with addition of cohensive ends of BamHⅠsite, taking pIRES-GHRH or pIRES-HBsAg-SS plasmid as template. The plasmid pCMV-Rep-GHRH or pCMV-Rep-HBsAg-SS were constructed by inserting a GHRH or HBsAg-SS fragment into the BamHI site of pCMV-Rep-LacZ replacing LacZ gene. Transforming to competent DH10B, recombinants were selected and identified by PCR, restriction enzyme digestion, orientation and DNA sequencing were carried out to verify the recombinant plasmids pCMV-Rep-GHRH and pCMV-Rep-HBsAg-SS. BamHⅠwas located at 3′end of GHRH gene in the pIRES-GHRH-HBsAg-SS plasmid. BamHⅠand BgLⅡare isocaudarner each other. We designed primers for GHRH gene, laying EocRⅠsite in the 5′end and BgLⅡsite in the 3′end of GHRH gene. GHRH gene fragment that obtained by PCR amplification, taking pIRES-GHRH plasmid as template was digested by EocRⅠand BgLⅡ, and then were ligated to pIRES-GHRH-HBsAg-SS with the same digestion and transformed to competent DH5α. The positive recombinants were selected and identified by PCR, restriction enzyme digestion and sequencing. After identification GHRH-HBsAg-SS gene fragment was obtained by PCR amplification, taking pIRES-GHRH plasmid mutated as template with upstream primer of GHRH gene and downstream primer of HBsAg-SS gene. pCMV-Rep-GHRH-HBsAg-SS was constructed by inserting a GHRH-HBsAg-SS fragment into the BamHI site of pCMV-Rep-LacZ replacing LacZ gene. PCR, restriction enzyme digestion, orientation and DNA sequencing were carried out to verify the recombinant plasmids pCMV-Rep-GHRH-HBsAg-SS. We have demonstrated that RNA replicon-based GHRH and SS co-expression in eucaryotic cell. The three expression vectors were transfected to prepared 293 and BHK-21 cells in 80% confluent according to provided protocols by Lipofectin. Results of RT-PCR, Tricine-SDS-PAGE, Western Blot and immunofluorescence antibody assay showed the our interested gene was expressed efficiently in transfected cell. The molecular weight of interest protein were match with those of expectation. The results indicated that SS antigen determinant were exposed in the surface of fusion protein by ELISA and RIA, possessing immunogenicity. The result confirmed that RNA replicon may significantly promote gene expression. To evaluate the effects of the SFV RNA replicon on the efficiency of gene expression, LacZ gene was inserted into pIRES which digested by BamHⅠand dephosphorylated by shrimp alkaline phosphatase, creating pIRES-LacZ in the present study. RNA replicon vector pCMV-Rep-LacZ and two conventional CMV promoter-based vector pLNCX-LacZ, pIRES-LacZ were transfected to prepared cells and mice muscles respectively. RNA replicon vector pCMV-Rep-GHRH and two conventional CMV promoter-based vector pcDNA3.1-GHRH, pIRES-GHRH were transfected respectively too. RNA replicon vector pCMV-Rep-HBsAg-SS and the conventional CMV promoter-based vector pIRES-HBsAg-SS were transfected respectively too. Results of quantitating forβ-gal expressed from transfected cells, quantitating for GHRH expressed from transfected cells by RIA and RT-PCR and for SS fusion protein expressed from transfected cells by RIA, RT-PCR and RT-Real Time PCR showed that the express level of RNA replicon vector was superior to ordinary vector and 3 times as high as that of two conventional DNA vectors. The concentration of serum HBsAb, serum IGF-I and anti-SS from transfected mice were detected by RIA and ELISA in this study. The results showed that the seurm antibody level and concentration of IGF-I from mice transfected RNA replion were higher signifiantly than that from ordinary vector respectively, however growth gain were not significantly higher than those from ordinary vector.We have demonstrated that the enhanced gene expression, genetic immunizationand and ability protecting plasmid DNA from degraded by nuclease due to microspheres adsorption. PLGA microspheres were prepared by double emulsion-in liquid evaporation process, using biodegrable ploy lactic-co-glycolic acid as carrier. The mean particle size were 10.58μm.. PLGA-MP-DNA compounds were prepared by mingling between PLGA-MP and DNA plasmid in proportion, incubated for 8~10 h at 37℃. The result that experiment for DNA protection by DNaseⅠdigestion and DNA immobilisation ability confirmed PLGA-MP-DNA compounds were able to adsorp and protect DNA plasmid. The result of mice muscular transfection in vivo showed that accumulative weight gain and concentration of serum IGF-I in the group injected with pCMV-Rep-GHRH-HBsAg-SS microspheres(40μg DNA) was significantly higher by 12.26%(P<0.01) than those groups injected with bare plasmid 35 days after injection. The anti-SS level in the group injected with plasmid microspheres were significantly higher by 10.36%(P<0.05) and 34.43%(P<0.05) than those groups injected with bare plasmid 14, 21 days after injection, respectively.The result confirmed that promoted growth and enhanced immunity in animal due to GHRH and HBsAg-SS gene transfer mediated by PLGA-MP. Mice were treated with pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP(40μg plasmid)and saline-MP by muscular injection, respectively. Immunohistochemistry from muscle injected site were detected. The result confirmed that interest protein corresponding were expressed in the mice muscle. After 35 days, the accumulative weight gain in those groups treated with pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP were 20.69~37.27%(P<0.01)significantly higher than the group injected with saline-MP. After 3~35 days, Concentrations of serum IGF-I in those groups treated with pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP were higher than the group injected with saline-MP, the best one was 143.49%(P<0.01) significantly higher than control group. The result of ELISA and RIA detection for serological specificity HBsAb and anti-SS were positive. The growing-finishing pigs were treated with pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP(1.5 mg plasmid)and saline-MP by muscular injection, respectively. After 120 days, the accumulative weight gain in those groups treated with pCMV-Rep-HBsAg-SS-MP were 6.36%(P<0.01)significantly higher than the group injected with saline-MP. The result of ELISA and RIA detection for serological specificity HBsAb and anti-SS were positive after injected 2 week. After 14~64 days concentrations of serum IGF-I in the groups treated with plasmid MP were significantly 11.41~66.34%(P<0.01) higher than control group, one of serum GH were higher 12.49~48.68%(P<0.01), and serum SS were lower 12.49~48.68%(P<0.05). The result that lymphocyte stimulation indices and the level of swine pest antibody detected confirmed GHRH and HBsAg-SS gene transfer could improve immunity in growing-finishing pigs.We have also demonstrated that promoted growth and enhanced immunity in first-generation progeny due to GHRH and HBsAg-SS gene transfer mediated by PLGA-MP. Pregnant rat dams were injected intramuscularly at day 14 d of gestation with 100μg of pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP. The result showed the born weight were significantly higher in the group treated with pCMV-Rep-HBsAg-SS-MP than the group injected with saline-MP. At day 35 after injected weight gain folds were 8.48%(P<0.05)~25.86%(P<0.01), concentrations of serum IGF-I were 31.34~56.19%(P<0.01)significantly higher in two group treatment group than control group, and serum SS in the groups treated with pCMV-Rep-HBsAg-SS-MP were lower 36.24%(P<0.01). The proportion of GH-positive cells was 43.39%~61.46%(P<0.01) significantly increased in teatment group than the control group. Gilts were injected intramuscularly at day 85~90 of gestation with 1 or 5 mg of pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP. Piglets were weighed from birth to weaning and bled at weaning. The result showed the weight of born, 20 d and 50 d were significantly 20.32~110.76%(P<0.01)higher than the group injected with saline-MP. After 50 days, concentrations of serum IGF-I in the groups treatmet groups with plasmid were significantly 2~3 times(P<0.01) higher than the group injected with saline-MP, one of serum GH were higher 11~26% (P<0.01).The result that lymphocyte stimulation indices and the level of swine pest antibody detected confirmed GHRH and HBsAg-SS gene transfer could improve immunity in first-generation progeny pigs.The result confirmed that a synergistic action displayed between GHRH and SS from co-expression vector pCMV-Rep-GHRH-HBsAg-SS in growth-enchancing for animal. The results from experiment in mice showed that the accumulative weight gain in the groups treated with pCMV-Rep-GHRH-HBsAg-SS-MP were 12.80~13.74%(P<0.01)significantly higher than those group injected with pCMV-Rep-GHRH-MP and pCMV-Rep-HBsAg-SS-MP at day 35 after injected. At 3 and 35 day, concentrations of serum IGF-I in the groups treated with pCMV-Rep-GHRH-HBsAg-SS-MP were 29.19~141.26%(P<0.05) significantly higher than injected with single gene plasimd group. The results from experiment in first-generation progeny mice showed that weight in double genes groups were 5.02(P>0.05)~16.02%(P<0.05)and the proportion of GH-positive cells was 9.09~13.01%(P<0.05)significantly highter in teatment group than single gene group after 59 d born. Gilts were injected intramuscularly at day 85~90 of gestation with 5 mg of pCMV-Rep-GHRH-HBsAg-SS-MP. Weight at weaning, serum IGF-I and GH, lymphocyte stimulation indices and the level of swine pest antibody were significantly higher than single gene group. The results from this study substantiated that GHRH and SS co-expression displayed synergistic action about growth-enchancing and improve immunity in mice and progeny pigs.更多还原