title: Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain.
abstract: Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the 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 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3h post-challenge. After a decrease at 6h, the expression level increased again and reached 207.8-fold at 12h post-challenge. 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) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (Ki) of rCfKZSPI-12 to trypsin was 173 nmol L(-1). These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response.
authors: Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China; Graduate School, Chinese Academy of Sciences, Beijing 100049, China.
authors: Bo
authors: B
authors: Wang
authors: Jianmin
authors: J
authors: Zhao
authors: Linsheng
authors: L
authors: Song
authors: Huan
authors: H
authors: Zhang
authors: Lingling
authors: L
authors: Wang
authors: Chenghua
authors: C
authors: Li
authors: Peilin
authors: P
authors: Zheng
authors: Ling
authors: L
authors: Zhu
authors: Limei
authors: L
authors: Qiu
authors: Kezhi
authors: K
authors: Xing
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D005786:Gene Expression Regulation; D006434:Hemocytes / Q000378:metabolism / Q000382:microbiology; D007700:Kinetics; D008969:Molecular Sequence Data; D049897:Pectinidae / Q000235:genetics* / Q000378:metabolism* / Q000382:microbiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors; D014733:Vibrio / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors