| Somatostatin(SS) is a short peptide of 14 amino acids inhibitory to the release of growth hormone. The SS gene was fused to 3' end of hepatitis B surface antigen (HBsAg) gene as HBSS gene. The HBSS fusion gene was cloned into pUC18. PCR amplification, restriction enzyme digestion and sequencing analysis confirm the successful construction of the recombinant pUC18-HBSS plasmid. The HBSS gene was cloned into the procaryotic expression plasmid pGEX-6p-l as pGEX-HBSS, which was then transformed into the E.coli BL21. The positive clones were confirmed by PCR and restriction enzyme digestion. The fusion protein was expressed upon induction with EPTG as identified by SDS-PAGE and radio-immunoassay. The target protein was about 20% of total extracable of E.coli BL21 and was reactive to SS antibody.The HBSS fusion gene was cloned into the eukaryotic plasmid pcDNA3 as pcDNA3-HBSS. The recombinant pcDNA3-HBSS plasmid was transformed by electroporation into an attenuated Salmonella typhimurium ZJ111 (dam~- and phop~-), resulting in ZJ111/pcDNA3-HBSS. The recombinant plasmid in the strain ZJ111/ pcDNA3-HBSS was identified by PCR and restriction enzyme digestion. The strain ZJ111/ pcDNA3-HBSS was subcultured for 10 times on Amp~- and Amp~+ LB agar plates for analysis of the in vitro stability of the recombinant plasmid within the bacterial cells. ZJ111/ pcDNA3-HBSS was used to infect the HeLa cells. The total RNA was extracted from the cells 48h post infection for RT-PCR analysis of mRNA transcription, SDS-PAGE and radio-immunoassay of the cell lysate to indicate the expression of the target gene. The result showed the mRNA of target gene was verified by RT-PCR and the target protein was reactive to SS antibody.The strains ZJ111/pcDNA3-HBSS and ZJ111/pcDNA3 were cultured in Amp~+ LB at 37℃ for 10h. In vivo infectivity of ZJ111/pcDNA3-HBSS and stability of the recombinant plasmid were tested by feeding the strain to grass carps through oral route. The bacterial isolates from the livers and spleens of fishes taken 7 days post-inoculation were identified by biochemical tests and PCR specific for S. typhimurium. The target genefrom pcDNA3-HBSS extracted from the in vivo bacterial isolates was verified by PCR and restriction analysis. The results indicate that the strain ZJlll/pcDNA3-HBSS could invade into the grass carps and the recombinant plasmid was stable in the host bactreria from the liver and spleen. The juvenile grass carps were fed with the strain ZJlll/pcDNA3-HBSS( 108cfu/ml,109cfu/ml, 1010cfu/ml)through oral route for evation of its safety to fishes, in comparison with wild style S.typhimurium (109cfu/ml) and PBS as controls. In another experimental setting, larger grass carps (body weight at about 150g) fed with 1 ml of ZJlll/pcDNA3-HBSS (108cfu/ml), S. typhimurium (108cfu/ml) and PBS. The livers and spleens were taken 7 days post-feeding for electronic microscopic analyses. Mortalities of juvenile grass carps fed the strain ZJlll/pcDNA3-SS at 108cfu/ml, 109cfu/ml and 10l0cfu/ml were 0%, 6.67% and 26.67% respectively. The cells of livers and spleens were morphologically normal from the fishes fed with ZJlll/pcDNA3-HBSS and PBS, while there were apoptotic changes of liver and spleen cells from fishes fed with wild type S. typhimurium. These results suggest that oral administration of ZJlll/pcDNA3-HBSS at the dose of 108 cfu/ml was relatively safe for carps.The enhanced green fluorescent protein(eGFP) gene was cloned into the eukaryotic plasmid pcDNA3 as pcDNA3-eGFP. The recombinant plasmid pcDNA3-eGFP was transformed by electroporation into attenuated Salmonella typhimurium (ZJ111), resulting in ZJlll/pcDNA3-eGFP. The postive clones for the strain ZJ111/ pcDNA3-eGFP were identified by PCR and restriction enzyme digestion. The juvenile grass carps were orally fed with ZJlll/pcDNA3-eGFP (108cfu/ml) to examine if eGFP gene could be expressed in the liver and spleen of grass carps. Flourescent microscopic examination revealed that eGFP gene was expressed in the liver and spleen of grass carps.The juvenile grass carps with the average body weight of 16g were fasted for one day and randomly divided into three groups(A,B and C),each for four replicates. Fishes in groups A and B were orally fed with ZJlll/pcDNA3-HBSS and ZJlll/pcDNA3 respectively at the dose of 108 cfu/ml, and fishes in group C fed with PBS. Four fishes from one replicate groups A and B were slaughtered 10 days post-feeding, and the liver and spleen samples were collected forpreparation of thin sections for immunohistochemistrical analyses. During the experimental period, the remaining fishes were fed with complete ration and grass in the water of dissolvable oxygen (^5mg/L), average temperature (25±2°C) and pH7.2 for 50 days. The body weight and length were recorded after the grass carps fasted for one day at the end of the experiment. Three fishes from each group were slaughtered and a piece of proximal intestines were takenfor electronic microscopic analyses. Blood samples were collected from the heart of the fishes of each group for determination of SS content. The sections of liver and spleen samples from the fishes fed with ZJlll/pcDNA3-HBSS was reactive to SS antibody 10 days post-feeding. Grass carps in group A had increased daily body gain as compared with those in groups B and C, but with no statistical difference. The length of micro villa of proximal intestines were 1.75(am, Llljim, 1.25u.m from fishes of groups A, B and C respectively. The SS content in the blood of fishes from group A was significantly lower than those of other groups. The above results indicate that HBSS protein expressed in the fish body reduced the SS content of the blood, but increased the length of micro villa and improved the growth performance of grass carps possibely through immuno-neutralization mechanism.In conclusion, the present study clearly indicates that the HBSS gene was expressed both in vitro and in vivo in grass carps. DNA vaccination mediated by attenuated S.typhimurium as oral delivery vector could improve the growth performance.
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