Molecular Cloning, Expression Of Serine Protein Inhibitor Genes CfKZSPI And Aikunitz From Scallops, And Inhibitory Activity Of The Recombinant Proteins

Scallop aquaculture is a big industry and contributes enormously to the economic development of coastal provinces in China. Since the summer of 1997, large-scale mortality of cultured scallop has caused catastrophic losses to scallop aquaculture, which resulted in the drastic decrease of the production. Serine proteases inhibitors (SPI) together with serine proteases play a central role in the invertebrate immune response. They are mainly involved in signaling and amplification cascades that lead to the activation of some defense processes, such as melanization, coagulation and induction of antimicrobial peptides. Therefore, the cloning, expression and functional study of the two SPI genes from scallop will help us to understand the scallop's immune defense mechanisms, and provide new information for the development of marine invertebrate immunology.A novel Kazal-type SPI gene was cloned from Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 bp, consisted of a 5'untranslated region (UTR) of 97 bp, a 3'UTR of 161 bp including canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1530 bp encoding a polypeptide of 509 amino acids. The deduced amino acid sequence of CfKZSPI contained a putative signal peptide of 22 amino acids and twelve tandem Kazal domains. The temporal expression of CfKZSPI in haemocytes after Listonella anguillarum challenge was recorded by quantitative real time PCR (QRT-PCR). The expression of CfKZSPI was up-regulated to 43.6-fold against the blank at 3 h post-challenge, decreased to 15.1-fold at 6 h, and then increased again and reached 174.1-fold, 207.8-fold and 675.4-fold at 8 h, 12 h, 24 h, respectively. The statistical analysis revealed that the expression level of CfKZSPI at 3 h and 12 h was significantly higher than that in the blank group. These results indicated that CfKZSPI was involved in the immune response in Zhikong scallop. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCfKZSPI-12) displayed significant inhibitory activity against trypsin, while no activity against thrombin. Through the method of Dixon plot the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was obtained to be 173 nmol L-1.A Kuintz-type SPI (Aikunitz) was cloned from the cDNA library of Bay scallop Argopecten irradians with the same method. The full-length cDNA of Aikunitz gene was of 632 bp, consisted of a 5'UTR of 105 bp, a 3'UTR of 245 bp and an ORF of 282 bp encoding a polypeptide of 93 amino acids. The deduced amino acid sequence was consisted of a signal peptide of 20 amino acids and a Kunitz domain. The temporal expression profiles of Aikunitz in haemocytes after Listonella anguillarum and Micrococcus luteus challenge were recorded by QRT-PCR. The mRNA level after Listonella anguillarum challenge was up-regulated from 3h to 9h, and reached 4.49-fold (P=0.008<0.05) against the control at 9h, then down-regulated, and reached 0.24-fold (P=0.021<0.05) against the control at 72h. The mRNA level after Listonella anguillarum challenge was up-regulated from 3h to 12h, and reached 5.95-fold (P=0.0004<0.01) against the control at 6h, then down-regulated rapidly after 12h, and reached 0.38-fold against the control at 24h. Aikunitz was recombined and expressed to investigate its inhibitory activity. The purified recombinant protein (rAikunitz) did not display inhibitory activity against the two potential serine proteinases, trypsin and elastase, nor to the growth of Gram-positive bacteria Micrococcus luteus, or Gram-negative bacteria Listonella anguillarum and E.coli.