Molecular cloning,sequence analysis and expression pattern of β-defesin 1 in Megalobrama amblycephala
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摘要:
从团头鲂（Megalobrama amblycephala）中克隆出β-防御素1（maBD-1）基因，检测该基因在组织中的分布，及在病原菌感染后该基因表达量的变化情况。将maBD-1基因重组到pGEX-KG原核表达载体上，构建重组质粒pGEX-KG-maBD-1，重组质粒经转化至BL21感受态细胞中进行诱导表达，将融合蛋白免疫日本大耳白兔，制备该融合蛋白的多克隆抗体，并检测血清效价。 结果表明：maBD-1基因ORF全长为204 bp，编码67个氨基酸。该基因在组织中广泛分布，病原菌感染后头肾和脾脏组织maBD-1基因表达量显著上调。经间接酶联免疫分析（ELISA）获得的抗血清效价为1∶3 200，在肝脏、鳃和心脏组织中用Western blot 可检测到β-防御素的表达。
Abstract:
The maBD-1 cDNA partial sequence of Megalobrama amblycephala was cloned using RT-PCR,its distribution in different tissues and expression after pathogen invasion was investigated.The target gene was then ligated with the Plasmid pGEX-KG vector and the pGEX-KG-maBD-1 was transformed into the prokaryotic cell BL21 and expressed by chemical inducer.The polyclonal antibody of the fusion protein was acquired by the immunity to rabbit and the titer of the antibody was measured.The results showed that the length of maBD-1 open reading frame was 204bp,encoding 67 amino acids.The maBD-1 gene was widely expressed in many tissues,and the expression in the head kidney and spleen was upregulated along with the pathogen invasion.Western-bloting analysis showed that the antiserum had the ability to combine with the GST-maBD-1 protein.