The plant expression vector of choleratoxin B subunit（CTB）-human insulin（BA） fusion protein pBI121/（CTB-BA） was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express C...The plant expression vector of choleratoxin B subunit（CTB）-human insulin（BA） fusion protein pBI121/（CTB-BA） was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express CTB-human insulin fusion protein was constructed and its hypoglycemic effect was evaluated in mice. The plant expression vector pBl 121/（CTB-BA） was digested with both BamI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/（CTB-BA） via the freeze thawing method, then CTB-BA gene was integrated to G Pentapyhllum Makino callus cells by co-culturing the cells with the transformed LBA4404 strain. The transformed G Pentapyhllum Makino callus cells were identified by DNA sequence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice. The sequences of synthetic CTB and human insulin genes（BA） were completely identical to those designed. Restriction map proved that the length of gene fragment in- serted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed. The result of RT-PCR was consistent with that expected. The expressed product showed a specific band with a relative molecular mass of 17000 by Western-blot. The human insulin expression amount was 6.03 μIU/mL according to the ELISA result. The animal test showed that only the G Pentapyhllum Makino callus cell line itself showed activity in decreasing the blood sugar of mice, however, the activity of the transformed G Pentapyhllum Makino callus cells was much higher, The plant expression vector pBI121/（CTB-BA） was constructed and expressed in the G Pentapyhllum Makino callus cells successfully for the first time. The trans- formed G Pentapyhllum Makino callus cells showed high activity in decreasing the blood sugar of mice. This study developed a new way for the development of oral administration insulin.展开更多
The N-terminal signal sequence of glucitol pcrmease of Escherichia Coli (Gut22) and its analogue (Gut22Ana) were synthesized. The analogue had a Pro residue substituting for the His at the 7th position of Gut22 and a ...The N-terminal signal sequence of glucitol pcrmease of Escherichia Coli (Gut22) and its analogue (Gut22Ana) were synthesized. The analogue had a Pro residue substituting for the His at the 7th position of Gut22 and a Val residue substituting for the Glu at the 10th position. The intrinsic fluorescence emission spectra indicated that the binding of Gut22 with lipid bilayer was much stronger than that of Gut22Ana. The leakage experiments with calcein-loaded liposomes showed that Gut22 strongly perturbed lipid bilayers while Gut22Ana did not. The apparent partition constant of Gut22 for partitioning into phosphatidylserine/phosphatidylcholine bilayers was measured; the effect of membrane potential on the interaction of Gut22 with lipid bilayers was studied and the conformation changes of Gut22 and Gut22Ana upon interacting with liposomes were studied by the method of circular dichroism analysis.展开更多
基金Supported by the Science and Technology Development Planning Foundation of Jilin Province of China(No.20030405)the Party and Government Administration "First-Leader-Hand" Science and Technology Project of Yunnan Province of Chi-na(No.2008QA028)
文摘The plant expression vector of choleratoxin B subunit（CTB）-human insulin（BA） fusion protein pBI121/（CTB-BA） was constructed first and then the Gynostemma Pentapyhllum Makino callus cell line that could express CTB-human insulin fusion protein was constructed and its hypoglycemic effect was evaluated in mice. The plant expression vector pBl 121/（CTB-BA） was digested with both BamI and SacI. Agrobacterium tumerfaciens strain LBA4404 was transformed with previously constructed recombinant plasmid pBI121/（CTB-BA） via the freeze thawing method, then CTB-BA gene was integrated to G Pentapyhllum Makino callus cells by co-culturing the cells with the transformed LBA4404 strain. The transformed G Pentapyhllum Makino callus cells were identified by DNA sequence assey and RT-PCR. The expressed product was identified by western-blot and its amount was tested by ELISA kit and its blood sugar decreasing effect was tested in mice. The sequences of synthetic CTB and human insulin genes（BA） were completely identical to those designed. Restriction map proved that the length of gene fragment in- serted into expression vector pBI121 was consistent with that expected. The sequence of genomic DNA of expressed product was completely identical to that designed. The result of RT-PCR was consistent with that expected. The expressed product showed a specific band with a relative molecular mass of 17000 by Western-blot. The human insulin expression amount was 6.03 μIU/mL according to the ELISA result. The animal test showed that only the G Pentapyhllum Makino callus cell line itself showed activity in decreasing the blood sugar of mice, however, the activity of the transformed G Pentapyhllum Makino callus cells was much higher, The plant expression vector pBI121/（CTB-BA） was constructed and expressed in the G Pentapyhllum Makino callus cells successfully for the first time. The trans- formed G Pentapyhllum Makino callus cells showed high activity in decreasing the blood sugar of mice. This study developed a new way for the development of oral administration insulin.
基金Project supported by the National Natural Science Foundation of China.
文摘The N-terminal signal sequence of glucitol pcrmease of Escherichia Coli (Gut22) and its analogue (Gut22Ana) were synthesized. The analogue had a Pro residue substituting for the His at the 7th position of Gut22 and a Val residue substituting for the Glu at the 10th position. The intrinsic fluorescence emission spectra indicated that the binding of Gut22 with lipid bilayer was much stronger than that of Gut22Ana. The leakage experiments with calcein-loaded liposomes showed that Gut22 strongly perturbed lipid bilayers while Gut22Ana did not. The apparent partition constant of Gut22 for partitioning into phosphatidylserine/phosphatidylcholine bilayers was measured; the effect of membrane potential on the interaction of Gut22 with lipid bilayers was studied and the conformation changes of Gut22 and Gut22Ana upon interacting with liposomes were studied by the method of circular dichroism analysis.
