墨吉明对虾(Fenneropenaeus merguiensis)CrustinⅡ型基因的克隆与原核重组表达

Cloning and Prokaryotic Recombinant Expression of Crustin Ⅱ Typed Gene of Fenneropenaeus merguiensis

Crustin是一类具有广谱抗菌活性的且富含半胱氨酸的小分子抗菌肽,广泛分布于甲壳动物中。为研究CrustinⅡ基因在墨吉明对虾中的免疫功能,本研究采用了RT-PCR技术获得了墨吉明对虾CrustinⅡ基因的ORF序列,共390 bp,编码一个129个氨基酸残基的多肽,且具有典型的Ⅱ型Crustin基因结构：N-末端的信号肽、C-末端的WAP（whey acidic protein）结构域、Gly和Cys富集区;运用实时荧光定量PCR技术检测了CrustinⅡ基因在墨吉明对虾不同组织中的表达模式,数据分析表明,CrustinⅡ在血细胞中高量表达,而在肠道、鳃、肝胰腺、尾部肌肉、胃、眼柄和心脏组织中的表达量非常低,且具有显著性差异;同时本研究运用原核重组表达技术成功诱导出CrustinⅡ蛋白。本研究为进一步探究Crustin在对虾的先天性免疫功能和防御机制提供理论依据。

Crustin is a kind of micromolecule antibacterial peptide with broad-spectrum antimicrobial activity and rich cysteine which widely spreads in crustaceans. To study the immune functions of Crustin Ⅱ gene in Fenneropenaeus merguiensis, the Open Reading Frame（ORF） sequence of Crustin Ⅱ gene was obtained by using RT-PCR and its length was 390 bp, which encoded 129 amino acids and possessed the typical type Ⅱ Crustin gene structures, including signal peptide in N-terminal, WAP structure of the domain in C-terminal, Gly and Cys enrichment region. The expression of Crustin Ⅱ gene in different tissues was tested by Real-time PCR. The data analysis showed that Crustin Ⅱ gene was highly expressed in blood tissue, while was lowly expressed in such tissues as intestines, gill, hepatopancreas, muscle, stomach, eyestalk and heart; and the difference was obvious. At the same time, Crustin Ⅱ protein was successfully induced by prokaryotic recombinant expression technology.This study would provide the theory basis to the further research on the congenital immune function and defense mechanism of Crustin in the prawn.