中国明对虾溶菌酶基因克隆、重组表达与性质分析

Molecular Cloning, Recombinant Expression and Characterization of Lysozyme from Chinese Shrimp Fenneropenaeus chinensis

溶菌酶是机体先天免疫系统中一个重要的效应分子,参与机体多种免疫反应,在溶菌过程中形成一个水解体系,破坏和消除侵入体内的病原,从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因),该基因全长709bp,其完整的阅读框为477bp,编码158个氨基酸,前18个氨基酸(-1^-18)为信号肽,成熟肽由140个氨基酸组成(1-140aa),其分子量为16.2kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达,但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a(+)中,转化大肠杆菌BL21(DE3),再进行诱导表达和亲和纯化,得到了纯化的重组溶菌酶,并进行了抑菌活性检测。结果表明,重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强,最小抑菌浓度达到3.43μmol/L,但对革兰氏阴性菌抑制作用较小。上述结果表明,该溶菌酶作为一种重要的免疫效应分子,参与了对虾的免疫防御反应。

Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens, We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme （FcLyz in short）, The full length of the gene was of 709 bp, and the open reading frame （477 bp） encoded 158 amino acids. The predicted protein had a signal peptide （-1- -18 residue） and molecular weight of the mature protein （residue 1-140） was of 16,2 kD. A Lyz 1 domain （residue 1-130） in the lysozyme was found by SMART analysis. The results of semiquantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp, The DNA fragment of mature Fc-Lys was subcloned to pET-30a （＋） expression vector, the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） and then induced by isopropylthio-β-D-galactoside （IPTG）, The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration （MIC） was assayed, Therecombinant protein showed high antibacterial activity against some Gram-positive bacteria, and MIC reached 3.43 μmol/L, and relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important molecules against pathogens in innate immunity of the shrimp.