Study On Molecular Characteristics And Function Of Cyclophilins In Freshwater Pearl Mussel, Hyriopsis Schlegelii

Hyriopsis schlegelii, which was originated in Lake Biwa of Japan, has better disease resistance than other freshwater pearl mussels. In this study, the CyPC, CyPD and CyPH cDNA full-length genes were obtained from freshwater mussels, Hyriopsis schlegelii using rapid amplification of cDNA ends PCR(RACE)technology. The fulllength cDNA sequence of HsCyPD was 2671 bp(GenBank No.:KJ747387), 5’-untranslated region(UTR) was 50 bp, 3’- UTR was 1487 bp with a poly adenosine signal(AAATAA) and a poly(A) tail, the open reading frame was 1104 bp encoded367 amino acids. The predicted molecular weight of the HsCyPD was 41.09 kDa and the theoretical isoelectric point(pI) was 5.43; The full-length cDNA sequence of HsCyC was 2050 bp, 5’- untranslated region(UTR) was 89 bp, 3’- UTR was 500 bp with a poly adenosine signal(AAAATAA) and a poly(A) tail, the open reading frame was 1461 bp encoded 486 amino acids. The predicted molecular weight of the HsCyPC was 55.93 kDa and the theoretical isoelectric point was(pI) was 6.07; The full-length cDNA sequence of HsCyPH was 761 bp(GenBank No.:KJ747386), 5’-untranslated region(UTR) was 70 bp, 3’- UTR was 151 bp with a poly adenosine signal(AAATAA) and a poly(A) tail, the open reading frame was 540 bp encoded179 amino acids. The predicted molecular weight of the HsCyPH was 19.66 kDa and the theoretical isoelectric point(pI) was 7.60. The bioinformatics analysis showed that CyPD, CyPC and CyPH with the typical peptidyl-prolyl cis-trans isomerases protein functional domains of cyclophilin;Meanwhlie, the system of evolutionary tree showed that the evolutionary status was quite conservative of CyPD, CyPC and CyPH.With the complete ORF of CyPB gene and vector pET-32 a, we constructed a recombinant expression vector pET-32a-CyPB, and recombinant plasmid transformed into BL21(DE3) for Prokaryotic expression, the amolecular weight about 40 kda protein was successfully expressed in the supernatant by IPTG induction, then the supernatant were puri?ed and obtained by using nickel ion affinity chromatography.Japanese white rabbits were immuned with purified CyPB and the polyclonal antibody which had a high-titer up to 1:512000 were obtained and western blot results showed that the polyclonal antibody had good specificity. Then we conducted liver frozen sections of Hyriopsis schlegeli for immunofluorescence localization using the CyPB polyclonal antibody, results showed that HsCyPB protein were expressed in the cytoplasm and cell membrane of liver cells.The fluorescent real-time quantitative PCR results showed that CyPA and CyPB were expressed in the eight tissues of Hyriopsis schlegelii,and the expression level in liver was the highest, the moderate expression was intestines and gonads, the lowest expression level was in the hemolymph. The complete ORF of HsCyPA and HsCyPB genes were subcloned into pEGFP-C1 eukaryotic expression vector; In the process of transfection experiment, Human liver cancer cells(HePG2) was used as experimental cells, experimental group of A, B, C, D represented pEGFP-C1, pEGFP C1-CyPA,pEGFP-C1-CyPB, pEGFP-C1-CyPA + pEGFP-C1-CyPB respectively. The control group and experimental group plasmids were transfected into human liver cancer cells(HePG2) using transfection reagents for eukaryotic expression. After growing 36 h, we found that the experiental group fluorescent level was obviously stronger than the control group under the check of confocal microscope. Meanwhlie, the similar results were found using flow cytometry. All these results indicated that HsCyPA and HsCyPB could promote the proliferation of human liver cancer cells(HePG2).