Study On Molecular Characteristics Of Cyps (Cyclophilins) And Preliminary Function Of CypA, Hyriopsis Schlegelii

Taking a high quality freshwater pearl calm, Hyriopsis schlegelii as research subject, this study introduced the process of using RACE PCR cloning technology to obtain the CypB, CypD and Cypl8.9full-length genes. Our results show that the Cyps of Hyriopsis schlegelii included CypB, CypD, Cypl8.9and CypA (Genbank login number:HM027883).CypB, whose open reading frame was encoding210Amino acid, was composed of944nucleic acids. the molecular weight of CypB protein was22.98kDa, and the theory isoelectric point(PI) was9.10. CypB was composed of739nucleic acids, and the open reading frame was between140and612nucleosides, encoding124Amino Acids. Its molecular weight was14.2kDa, and the theory isoelectric point was9.67. Cyp17.89had1184nucleic acids, but only encoded169Amino Acids, and the molecular weight of CypB was17.89kDa, and its PI was5.49. The biological information analysis showed that CypB had a strong tail signal, composed of19-22Amino Acids. The3-D structure of Cyps was like a right hand barrel that formed by eight reverse parallel folding strands. Its top and bottom connected to some complex stem ring (loop) structures and the β-strands, which connected to3a-helix on both ends. The3-D structure of CypA, CypB, CypD was similar, but slightly different from Cyp18.9. To identify the evolutionary relationship of Cyps, the system evolutionary tree structure analysis was performed. The result show that the closest Cyps kinship relations of Hyriopsis schlegelii is Conus novaehollandiae.CypA, whose molecular characteristics of gene expression and Immunological stress function under duress of pathogenic bacteria had been confirmed, was the first Cyps member found in blood cells cDNA library of Hyriopsis schlegelii. The polyclonal antiserum had been prepared before(unpublished). To identify the special combination beteewn the polyclonal antiserum and CypA from recombinant, liver and gonad tissues, Western Blot Technique was employed. It showed that there were very bright stripes in18kDa and37kDa. To examine the stimulated effect of recombinant protein of prokaryotic expression to Hela cell line, the CypA would be performed on corresponding concentrations (0,50,100,200,400,800ng/ml). The study revealed that, to some extent, the CypA can promote the proliferation of Hela cells, and the optimum concentration is approximate100ng/ml.