ABSTRACT
OBJECTIVE To optimite the purification technology of total triterpenoid extracts from Inonotus obliquus ，and to investigate the anti -tumor activity of its purified products . METHODS Using inotodiol as control ，the method was established for the content determination of total triterpenoid in I. obliquus. The type of macroporous adsorption resin ，sample volume ，sample concentration，sample flow rate ，eluent volume ，eluent dosage and elution flow rate were selected by single factor experiments . The purification technology of the crude extract was determined and verified . The effects of total triterpenoid purified from I. obliquus on the proliferation ，migration and apoptosis of human cervical cancer HeLa cells were detected by cell proliferation test ， migration test ，flow cytometry and AO/EB kit . RESULTS The best purification technology of total triterpenoid crude extracts from I. obliquus was as follows ：AB-8 macroporous adsorption resin was used ；mass concentration of the sample solution was 2.0 mg/mL；sample volume was 140 mL，and the flow rate was 1.0 mL/min；the impurity was removed with 50% ethanol 40 mL， then eluted with 95% ethanol 160 mL，at the elution flow rate of 3.0 mL/min. After purification ，mass concentration of total triterpenoid from I. obliquus increased from 34.36% to 73.39%. The total triterpenoid of I. obliquus could inhibit the proliferation of HeLa cells ，and the 50% inhibitory concentration was 184.20 μg/mL. Compared with control group ，the purified products could significantly inhibit the migratio n and promote the apoptosis of HeLa cells （P＜0.05 or P＜0.01）. CONCLUSIONS The purification technology of total triterpenoids extracts from I. obliquus is successfully optimited . The purified product could inhibit the proliferation and migration of HeLa cells and induce their apoptosis.
ABSTRACT
Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.
ABSTRACT
Objective: To construct mcpr1prokaryoti c expression vector and to express MCPR1 protein.Methods:PCR was used to obtain coding region of mcpr1. Construction of a high-level fusion protein expression vector pGEX-4T-mcpr1 was conducted by inserting the fra gment of coding region of mcpr1into a fusion protein expression vector pGEX -4T-1. Then the recombinant plasmid was transferred into E. colito prepar e the MCPR1/GST fusion protein. DNA sequencing and endonucleases digesting were used to check the coding region. Results:pGEX-4T- mcpr1 wa s constructed successfully and the coding region was inserted into the vector co rrectly. A new protein band of 36 000 was observed by SDS-PAGE analysis after i nduction by IPTG. The 36 000 protein amounted to 39 percent of the total prote in and existed mostly in precipitation of broken bacteria. Conclusion: MCPR1 protein can be expressed in E. coliexpression system and purif ied initially.