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Expression of CTB-human Insulin(BA) Fusion Protein in gynostemma pentapyhllum makino callus cells and Its Hypoglycemic Effect in Mice

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Chemical Research in Chinese Universities 2010年第5期26卷 773-779页

作者： WANg Ya-jun QI Feng-chun HAO Shu-mei WANg Chun-yi SUI Bo SHENg Jun College of Life Science Jilin University Changchun 130012 P R. China Changchun Institute of Biological Products Changchun 130062 P R. China Changchun University of Science and Technology Changchun 130022 P. R. China Changchun Keygen Biological Products Co. Ltd. Changchun 130021 P R. China Yunnan Agricultural University Kunming 650201 P. R. China

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 stu

关键词： Human insulin peptide without C pcptide Choleratoxin B subunit g pentapyhllum makino callus cells

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