Cloning And Analysis Of C-type Lectin Gene From Freshwater Pearl Mussel, Hyriopsis Schlegelii

Hyriopsis schlegelii，which was native to Japan，belonging to Hyriopsis ofUnionidae，is a new class of high-quality freshwater pearl mussels. However， due tothe recent expansion of the scale farming and destruction of breeding environment，disease problems had occurred frequently in Hyriopsis schlegelii，which caused aheavy economic losses to their breeding.In the present experiment，according to thepartial fragment of C-type lectin from laboratory transcriptome，and with the aid ofthe RACE PCR reaction， Real-time PCR and prokaryotic expression technologies toclone the full-length cDNA C-type lectin and make correlation analysis for itsexpression，providing the foundation for further research on immune mechanism ofHyriopsis schlegelii.Screened from the laboratory transcriptome,the full-length cDNA of C-typelectin from Hyriopsis schlegelii was first cloned by RACE. Results indicated that theC-type lectin cDNA of Hyriopsis schlegelii had a full length of1716bp，including a5’untranslated region (5’UTR) of92bp,a3’untranslated region (3’UTR) of898bp andan open reading frame (ORF) of726bp encoding241amino acids.Real-time PCR technology was applied to detect the expression of C-type lectinin different tissues. It turned out that it is expressed in hepatopancreas, hemolymph,mantle, gill, adductor muscle and gonad, in which the expression of hemolymph ishighest，expression of mantle, hepatopancreas, gill and adductor muscle is followed，and the expression level of gonad was the lowest.After LPS stimulation，with the aid of quantitative PCR technology to assay theexpression of C-type lectin in hepatopancreas and hemolymph and gonad.It wasfound that the expression of C-type lectin in hepatopancreas and hemolymph werehighest at about24h，72h after LPS challenge，expression level were respectively1.72-fold，1.39-fold compared with control groups，while the expression peak ofC-type lectin in gonad appeared in48h，expression level was3.40times that ofcontrol groups. pGEX-4T-1-CLEC expression vector was constructed by double digestionreaction，then transformed into the expression bacteria Rosetta，finally fusion proteinwas successfully induced in0.5mM IPTG.Since the fusion protein existed in the formof inclusion bodies, then denaturating,refolding them,finally target protein waspurified by Glutathione Sepharose4B.