Bioinformatic Analysis, Recombination And Expression Of HSP20 From Echinococcus Multilocularis As Well As The Culture Of Alveolar Hydatid In Vitro

Objective:The aim of the present work was to predict the structure and function of Echinococcus multilocularis heat shock protein 20(EmHSP20) using bioinformatic methods and to clone and express EmHSP20 in E.coli prokaryotic expression system.Methods:The structure and function of EmHSP20 was analysed using various bioinformatic softwares. The coding sequence of EmHSP20 was subcloned into the plasmid pET-28b(+). The recombinant plasmid was transformed into DH5a and subsequently digested with Ndel and Xhol and sequenced to identify the inserted sequence. After that, the recombinant was transformed into Rosetta(DE3) and expressed in induction. EmHSP20 was purified with Ni-NTA column and analysed with western blot and SDS-PAGE. Finally, determinated the concentration of the recombinant protein.Results:EmHSP20 was a full-length sequence (1261 bp) with a coding sequence of 945 bp and 314 amino acids, corresponding to a deduced molecular mass of 35843.7 Da and isoelectric point of 5.92. EmHSP20 was an intracellular protein with no signal peptide and transmembrane region. EmHSP20 showed a characteristic duplicated alpha-crystallin domain and four kinds of sites, which mainly were phosphorylation sites. That suggested phosphorylation maybe a crucial post-translational modification of EmHSP20. EmHSP20 can combine with a variety of proteins with 8 protein binding sites. The recombinant plasmid pET-28b(+)-EmHSP20 was sucessfully constructed and EmHSP20 was effectively expressed in prokaryotic expression system. Both western blot analysis and SDS-PAGE analysis of the recombinant protein showed good results. The concentration determinated of EmHSP20 was 1.1mg/mL.Conclusion:We obtained the basic information of EmHSP20 through bioinformatics methods. pET-28b(+)-EmHSP20 recombinant plasmid was sucessfully constructed and efficiently expressed in prokaryotic expression system which laid the foundation for the following study of its immune protection and biological function.Objective: Culture alveolar hydatid with different mediums and serums to find more suitable condition to cuture alveolar hydatid in vitro.Methods: Alveolar hydatid was cultured respectively with DMEM, RPMI1640 and the serum of fetal bovine, mice and horse in vitro. Recorded their growth and observed the morphology and ultrastructure of vesicles,to find better condition to culture alveolar hydatid.Results: Among all culture conditions, alveolar hydatid cultured with DMEM-mice serum grew fastest, with 34 vesicels.There were 26 vesicels cultured with DMEM-fetal bovine serum, 13 vesicels cultured with RPMI1640-mice serum and 9 vesicels cultured with RPMI1640-fetal bovine semm.No vesicel was found in the culture of DMEM-horse serum or RPMI1640-horse semm.The size of the vesicles cultured by mice serum were smaller than the vesicles cultured by fetal bovine serum on the whole. There were no difference between four kinds of vesicles in ultrastructure under transmission electron microscope.Conclusion: The culture in vitro showed that mice serum was more suitable for the growth of alveolar hydatid than conventional fetal bovine serum and horse serum, and that DMEM was more suitable than RPMII640.