Molecular Cloning And Expression Of Kunitz And Serpin B Gene From Cristaria Plicata

Kunitz gene and Serpin B gene was cloned from Cristaria plicata, and analyzed their homology and tertiary structure. The expression of genes were deteced by quantitative PCR method in haemocytes, mantle, gill, muscle and hepatopancreas tissue of C. plicata after injected PBS, gram-negative bacteria Aeromonas hydrophila and LPS. Recombinant plasmid was obtained by enzyme digestion and purified protein after induction.The full-length cDNA sequence of Kunitz gene was 1378 bp,including a 5’-terminal untranslated region(UTR) of 128 bp, a 3’- terminal UTR of 482 bp and a poly(A) tail. The open reading frame was 768 bp, and encoded a polypeptide of 255 amino acids. The N-terminus had the features consistent with a signal peptide as defined by SignalP 4.0 software with a putative cleavage site located in position 17(MSTFFMFMYVSPHWYLQ), mature peptide consists of 239 amino acids. The protein isoelectric point is 8.32, and the molecular weight is 28.7 kD. The sequence is highly similar with Solen grandis, about 98%. Kunitz has four conserved sequence, including three α- helices and six β- sheet.The expression level of Kunitz gene in haemocytes, mantle, gill, muscle and hepatopancreas from Kunitz was estimated by semi-quantiative RT-PCR technology.The results showed that Kunitz expressed in each of tissues and the expression level of mantle was the minimum. While haemocytes and hepatopancreas had highest expression. After injected by A. hydrophila(109 cell/mL), the expression level of Kunitz in hepatopancreas and haemocytes increased, then reached its vertex at 12 h. Kunitz in haemocytes then reached its minimum at 48 h, while the expression level was higher in hepatopancreas. After injected by LPS, the expression level of Kunitz in hepatopancreas then haemocytes also reached its vertex at 12 h afterwards decreased,but the reaction of the hepatopancreas was more sensitive. In order to further inquire into the functions of Kunitz, to build recombinant plasmid with vector and the purpose gene. It was found that the recombinant plasmid established by the plasmid pET-32 and Kunitz after broken was appeared in the supernatant with soluble protein. The purification soluble protein of Kunit was about 40 kD, which was got high purity recombinant pET32-Kunitz and was confirmed by the analysis of the recombinant protein. It was identified with expectations.The full-length cDNA sequence of Serpin B gene was 1565 bp, including a 5’-terminal untranslated region(UTR) of 21 bp, a 3’- terminal UTR of 362 bp and a poly(A) tail. The open reading frame was 1182 bp and encoded a polypeptide of 393 amino acids. The protein isoelectric point was 6.57, and the molecular weight was 44.2 kD. The sequence was highly similar with Crassostrea gigas, about 47%. The protein had a Serpin structure domain which located in 25-391 amino acid called surper family protein.The expression level of Serpin B gene in haemocytes, gill, mantle, muscle and hepatopancreas from Serpin B was estimated by semi-quantiative RT-PCR technology.The results showed that Serpin B expressed in each of tissues and the expression level of gill was the minimum. While haemocytes and hepatopancreas had highest expression about 6.62 time and 6.85 time of gill’. After injected by A. hydrophila(109 cell/m L), the expression level of Serpin B in hepatopancreas and haemocytes increased gently between 6 and 24 h, afterwards reached its vertex at 24 h, especially highlighted in hepatopancreas. After injected by LPS, the expression level of Kunitz in hepatopancreas reached its peak at 12 h, then got a lowest expression at 48 h. The expression level of Kunitz in haemocytes had no obvious tendency in 6~12 h,but reached its vertex at 24 h.