Clone And Expression Analysis Of HSP60and HSP70during The Regeneration Caudal Fin In Two Species Of Loaches

Heat shock proteins (HSPs) are a kind of an extremely conservative protein molecules, theseproteins,which can be induced, protect organism from heat injury etc. They have some factions, such asprotein folding and repairing, cell protection, anti-apoptosisn and diseases. As the most class of themolecular chaperones, the biology functions of Heat Shock Proteins are abroad, HSP60and HSP70arecrucial members of them. The researchers have studied its structure, and many of their biological functionswere found, but they are rarely mentioned in the tissue regeneration and fin regeneration.Yellow Loach and Dabryanus were used for research, which have high adaptability andnutritional value. The full-length sequences of HSP60and HSP70is cloned in two loaches by the RACE.We analysis the sequences structure. A pair of ORF primers is designed containing restriction sites to clonean open reading frame (ORF). We build recombinant expression Strains with pET-32a for inducing theexpression of recombinant protein. GAPDH as reference gene, we analysis the expression pattern of HSP60and HSP70in the process of two loaches’ caudal regeneration fin by using relative fluorescence Real-timePCR, based on the reference genes of GAPDH.According to the technology of RACE, The full-length sequences of HSP60and HSP70wereobtained in two loaches’caudal fin regeneration. The ORFs of HSP60gene (encoding576amino acids，moleculars weight61.051kD, the theoretical isoelectric point4.75) in two loaches are1728bp. Theirsequences contain GGM repeats of HSP60family and AAVEEGIVPGGG Signal tag. The homologousdegree and evolutionary were analysised，we found HSP60sequence is highly conserved. The ORF ofHSP70gene (encoding644amino acids, moleculars weight70.397kD, theoretical isoelectric point4.38) in two loaches are1932bp. Their sequences contain the cytoplasmic label EEVD of HSP70family and threetags. the degree and evolutionary were analysised，we found that the sequence of HSP70is highlyconserved. After Inducing expression of recombinant proteins, the molecular weight of two recombinantproteins from loach are78.9kD and88.3kD (including partial vector encoding protein) after SDS-PAGEelectrophoresis analysising. The optimal expression condition is37°C,1mmol/l IPTG, expressing6h, Aftergradient optimizing of the temperature and IPTG concentration. HSP60and HSP70are involved in theprocess of fin regeneration by The Relative fluorescence Real-time PCR. The functions of HSP60andHSP70should not only participate in the formation of epidermal cells in the cross-section and blastemaformation of the regeneration process but also work on damage protein folding, repairing andanti-apoptotic.