诺卡氏菌mce1A基因的克隆及重组表达

Cloning mce1A gene from Nocardia seriolae and its expression

诺卡氏菌(Nocardia seriolae)是近年来海淡水养殖鱼类频发慢性传染病的病原菌。该研究通过引物扩增得到全长1 254 bp、编码417个氨基酸的诺卡氏菌毒力因子mce1A基因的全序列。该基因编码蛋白质的分子量约为43.98 k D,理论等电点5.14,含有161个疏水性氨基酸,疏水性平均值为0.044,为疏水性蛋白,具有α-螺旋、β-折叠和无规则卷曲3种结构。经预测,Mce1A有MCE、DUF3407区域、OM_asym_Mla D、Mtu_fam_mce、Mla D 5个结构域,彼此交叠,含有公共区域。根据Mce1A氨基酸构建的系统进化树可以发现诺卡氏菌和新星诺卡氏菌的亲缘关系最近。构建p ET32a-mce1A重组质粒并转化至大肠埃希菌BL21中,得到的重组蛋白的相对分子量约63 k D。温度、IPTG浓度对重组蛋白表达量没有影响。该研究为进一步研究Mce1A的致病机制奠定了基础。

Nocardia seriolae,a bacterial fish pathogen of marine fish and freshwater fish,causing nocardiosis,can lead to mass loss of fish farming. The full-length（ 1 254 bp） of mce1 A c DNA in N. Seriolae was cloned in this study. The Mce1 A molecular weight was43. 98 k D; the theoretical isoelectric point and theoretical hydrophobic were 5. 14 and 0. 044,respectively. It contained 161 hydrophobic amino acids,as the hydrophobic proteins. The protein mainly consisted of random coil,and almost had no helix and folding.Sequence analysis indicates that it encoded 417 amino acids,and contained five relatively conservative domains（ MCE,DUF3407,OM_asym_Mla D,Mtu_fam_mce and Mla D domain）. These domains overlaped each other and contained public areas. The study constructs a phylogenetic tree based on Mce1 A c DNA which shows that the members of N. seriole are closely related to N. nova. We constructed a recombinant plasmid p ET32a-mce1 A and then transfered to E. coli BL21（ DE3） in order to obtain recombinant protein. The E. coli containing recombinant vector expressed protein of 63 k D after induction by IPTG. The results provide references for further study on pathogenic mechanism.