褶纹冠蚌α_2M基因的组织分布及原核表达

Tissues Distribution and Prokaryotic Expression of Alpha-2 Macroglobulin from Cristaria plicata

α2-巨球蛋白(alpha-2 Macrogloblin,α2M)是存在于无脊椎动物和脊椎动物血浆中的一类广谱性蛋白酶抑制因子。本文利用RT-PCR方法分析了α2M基因mRNA在褶纹冠蚌不同组织的表达及在嗜水汽单胞菌刺激后褶纹冠蚌血细胞中的表达变化。结果表明,α2M仅在血细胞中有表达,而在外套膜、闭壳肌、肝胰腺和鳃组织中均无表达。注射嗜水气单胞菌6h、12h、24h后,褶纹冠蚌血细胞中α2M的mRNA表达水平显著升高,表明α2M是褶纹冠蚌基础免疫系统中的重要组成部分。选择褶纹冠蚌α2M基因包含有受体结合区片段的第1369～1589氨基酸设计含有酶切位点的表达引物,构建重组表达质粒,经过IPTG诱导表达,利用SDS-PAGE分析表达产物。结果表明重组的α2M在大肠杆菌Escherichia coli Rosetta-gami(DE3)中获得了表达,产物为40.81KDa的融合蛋白。

Alpha-2 Macrogloblin is one of broad spectrum protease inhibitory factor, which consist in plasma of vertebrates and invertebrates. Distribution of α2-macroglobulin （α2M） in different tissue and various of expression of α2-macroglobulin （α2M） in hemocytes of Cristaria plicata after stimulated by Aeromonas hydrophila were studied using RT-PCR method in the paper. The result showed that the mRNA transcript of C. plicata α2M can not be detected in mantel, adductor muscle, hepatopancreas and gill except for hemocytes. After injecting by A. hydrophila 6 h, 12 h, 24 h, α2M expression level was increased significantly in hemocytes. It suggested that α2M was an important component part of C. plicata immune system. The expression primers were designed according to part of cDNA segment which contained the receptor-binding domain of 1369~1589 amino acid of C. plicata α2M. The constructed recombinant expression plasmids were induced by the chemical inducer IPTG. The expression products were analyzed by SDS-PAGE afterwards. The results indicated that recombination α2M protein was successfully expressed in Escherichia coli. The molecular weight of expression product was 40.81 KDa.