Wang Bo, Zhao Jianmin, Song Linsheng, Zhang Huan, Wang Lingling, Li Chenghua, Zheng Peilin, Zhu Ling, Qiu Limei, Xing Kezhi
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China; Graduate School, Chinese Academy of Sciences, Beijing 100049, China.
Fish Shellfish Immunol. 2008 May;24(5):629-37. doi: 10.1016/j.fsi.2008.01.017. Epub 2008 Feb 8.
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.
丝氨酸蛋白酶抑制剂（SPIs）在所有多细胞生物的宿主生理和免疫过程中发挥着重要作用。通过表达序列标签（EST）和cDNA末端快速扩增（RACE）方法，从栉孔扇贝（Chlamys farreri）中克隆到一个新的卡扎尔型SPI基因（命名为CfKZSPI）。CfKZSPI的全长cDNA为1788个核苷酸，具有典型的多聚腺苷酸化信号序列AATAAA和一个polyA尾，一个开放阅读框（ORF）编码一个由509个氨基酸组成的多肽，带有一个22个氨基酸的推定信号肽。CfKZSPI推导的氨基酸序列包含12个串联的卡扎尔结构域，与其他卡扎尔型SPIs具有高度相似性。通过定量实时RT-PCR记录了鳗弧菌（Vibrio anguillarum）攻击后CfKZSPI在血细胞中的时序表达。CfKZSPI的相对mRNA表达水平在攻击后3小时上调，达到43.6倍。在6小时下降后，表达水平再次升高，在攻击后12小时达到207.8倍。将CfKZSPI的第12个卡扎尔结构域重组到pET-32a(+)中，并在大肠杆菌Rosetta-gami (DE3)中表达，以研究其抑制活性。纯化的重组蛋白（rCfKZSPI-12）对胰蛋白酶显示出显著的抑制活性，但对凝血酶无活性。当抑制剂与胰蛋白酶的摩尔比达到1:1时，几乎90%的酶活性可被抑制，这表明一个rCfKZSPI-12分子能够抑制一个胰蛋白酶分子。用Dixon图进行动力学分析表明，rCfKZSPI-12对胰蛋白酶的抑制常数（Ki）为173 nmol L(-1)。这些结果表明，CfKZSPI是一种新型的卡扎尔型SPI，对胰蛋白酶具有显著的抑制活性，并且推测其参与了扇贝的免疫反应。
