微小牛蜱一个抗菌多肽编码基因的克隆和表达

Molecular cloning and analysis of antimicrobial peptide of Boophilus microplus

为了进行抗蜱及蜱传病疫苗的研究 ,本研究根据微小牛蜱巴西株报道的一种抗菌多肽核苷酸序列设计引物 ,从微小牛蜱中国安徽株克隆到该抗菌多肽基因 ,全长 383bp ,编码 110个氨基酸残基 ,该蛋白预测的分子量为 12 2ku ,等电点为 4 87。经同源性比较 ,该微小牛蜱巴西株抗菌多肽基因有 10 0 %的相同性。经RT_PCR分析表明 ,该基因在微小牛蜱卵、幼蜱、半饱血雌蜱、饱血雌蜱和雄蜱这几个阶段均有表达。将该基因亚克隆到 pET_2 8a(+)表达载体 ,转化BL2 1(DE3)宿主菌 ,经IPTG诱导 ,可成功表达。重组融合蛋白大小为 15ku左右 ,与预期大小一致。初步体外抗菌试验表明 ,重组蛋白具有一定的抗菌活性。Western_blot显示 。

To control tick and tick_borne diseases, on the basis of the lately nucleotide sequence of structural specially microplusin from Brazil B.microplus,a gene encoding antimicrobial peptide was isolated and cloned from B.microplus of Anhui,China.It is composed of 383 nucleotides and encoding 110 amino_acid residues,the deduced mass is 12.2 ku,isoelectric point is 4.87.The sequence has 100 % similarity from the microplusin of Brazil B.microplus.RT_PCR analysis suggests the antimicrobial peptide are expressed in the eggs,larvas,fed female adult,engorgement female adult and male adult ticks of B.microplus.After cleaving the signal peptide the residual was subcloned into expressing vector pET_28a(+),and expressed in E.coli BL21(DE3).The recombinant protein shows a single band by SDS_PAGE,the size is about 15 ku.Antimicrobial experimentation in vitro shows that it seems have antibacterial activity partially. Western blotting analysis suggests the rabbit anti_B.microplus salivary serum can recognize recombinant protein.