Gene Clone And Expression Of Heat Stress Proteins And A Related Protein From Roughskin Soulpin (Trachidermus Fasciatus)

HSPs are a family of highly conserved cellular proteins that play a key role in the immune responses. In this study, the fundamental immunity of roughskin sculpin (Trachidermus fasciatus) was investigated based on the cDNA amplification and protein expression of HSBPl, HSC70, GRP94 and HS6B. The main results are listed as below.Full length cDNA of HSBPl (1821bp) was cloned with the RACE-SMART technique. The cDNA contained an open reading frame (ORF) of 324bp which encoded a 78 (Mr8.55kDa) amino-acid polypeptide with isoelectric point (pI) 4.16. The homology of HSBP1 between roughskin sculpin and other species submitted to GenBank was analyzed. The similarity was 82.28%-98.70% between roughskin sculpin and other fishes, and it was 75.32%-79.22% among roughskin sculpin and amphibia, birds, mammals. In order to construct a prokaryotic expression of HSBP1 in Rosseta, the HSBPl gene was cloned into prokaryotic expression vector pET-30a(+). The recombinant vector, pET-30a(+)/HSBP1, was transformed into Rosseta and was induced to express protein by IPTG. Then the protein product was identified by SDS-PAGE. The result showed that about 20kDa protein which was bigger than prediction was strongly expressed in Rosseta. The recombinant protein was soluble and purified by High-Affinity Ni-IDA Resin.Complete HSC70 cDNA sequence 2138bp in length was cloned. It contained an ORF of 1947bp which encoded a 629 (Mr71.1kDa) amino-acid polypeptide with isoelectric point (pI) 5.27. Three tag sequences and cytoplasmic motif (EEVD) in the C-end of HSP70 family were found from the amino-acid sequence. The alignment result indicated that the similarity was 88.89%-97.09% between roughskin sculpin and other fishes, and it was 85.36%-93.98% among roughskin sculpin and amphibia, reptiles, birds, mammals. The prokaryotic expression of HSC70 was successfully constructed in E. coil BL21(DE3), and the E. coil BL21(DE3) with recombinant vector, pET-30a(+)/HSC70, was induced to express peotein by IPTG. Then the protein product was identified by SDS-PAGE. The result showed that a 76.4KkDa protein was strongly expressed in E. coil BL21(DE3). Most of the recombinant protein formed into inclusion body with induction by IPTG at 37℃for 4h, while partial of it was soluble with induction by IPTG at 25℃overnight. The recombinant protein was purified by High-Affinity Ni-IDA Resin.Full length cDNA of GRP94 (2736bp) was cloned with the same method. The cDNA contained an ORF of 2418bp which encoded a 806 (Mr92.5kDa) amino-acid polypeptide with isoelectric point (pI) 4.75. Five tag sequences and motif in the C-terminal of HSP90 family were found from the amino-acid sequence, and variable sites in GRP94 amino-acid sequence of roughskin sculpin were detected. The front 22 amino-acids was the putative signal peptide, and the mature peptide contained a HATP_ase domain. The similarity of amino-acid sequence was 83.58%-89.95% between roughskin sculpin and other fishes submitted to GenBank, and it was 77.87%-81.09% among roughskin sculpin and amphibia, birds, mammals. Sequences of GRP94 from fishes were a separate branch in the phylogenetic tree. The recombinant vector, pET-30a(+)/GRP94, was constructed, then transformed into E. coil BL21(DE3), and was induced to express protein by IPTG. The protein product was identified by SDS-PAGE. The result showed that a 98.2kDa protein was weakly expressed in E. coil BL21(DE3). But, there was little effect on the percentage of target protein with the treatment of different concentration of IPTG (0.5 mM and 1 mM), differrent temperatures and time (37℃,4h; 25℃, overnight; 16℃, overnight).With the same method, the whole HS6B cDNA sequence (1127bp) was cloned. The cDNA contained an ORF of 522bp which encoded a 174 (Mr19.04kDa) amino-acid polypeptide with isoelectric point (pI) 8.63. The putative mature peptide contained a RHOD domain. The alignment among roughskin sculpin and other species submitted to GenBank baesd on amino-acid sequence of HS6B showed that the similarity was lower than the other HSPs. The recombinant vector, pET-30a(+)/RHOD, was constructed, transformed into E. coil BL21(DE3), and was induced to express protein by IPTG. The protein product was identified by SDS-PAGE. The result showed that an 18.4kDa protein was weakly expressed in E. coil BL21(DE3). The recombinant protein was strongly expressed with induction at 25℃overnight. And the percentage of soluble protein increased greatly. The recombinant protein was purified by High-Affinity Ni-IDA Resin.Through the analysis of the cDNA sequence and protein expression of the four important immunity-related proteins, this work provides scientific data for the efficient captivity and breeding of roughskin sculpin, and gets the new insight for the fish disease control.