斑点叉尾鲖BPI1基因的原核表达及生物信息学分析  

The prokaryotic expression for BPI1 gene of channel catfish(Ictalurus punctatus) and its bioinformatics analysis

作　　者：王兴丽[1] 汪开毓[1,2] 陈德芳[3] 朱劼垚 杨倩[1] 贺扬[1] 王二龙[1] 

机构地区：[1]四川农业大学鱼病研究中心,四川成都611130 [2]四川农业大学动物疾病与人类健康四川省重点实验室,四川成都611130 [3]四川农业大学动物科技学院,四川成都611130

出　　处：《水产学报》2017年第1期1-10,共10页Journal of Fisheries of China

基　　金：四川省科技厅应用基础项目(2014JY0143);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848)~~

摘　　要：为探究斑点叉尾鲖BPI1抗菌肽基因编码蛋白的结构特征,实验提取斑点叉尾鲖肾脏RNA,根据BPI1基因序列设计引物,利用PCR方法克隆BPI1基因并构建至原核表达载体p TWIN1中。构建成功的重组质粒p TWIN1-BPI1经原核表达,SDS-PAGE检测显示得到约为50 ku的融合蛋白,与预期结果相一致。通过生物信息学软件对BPI1基因编码的蛋白序列进行分析,结果显示,克隆所得斑点叉尾鲖BPI1基因编码一条由223个氨基酸残基组成的多肽,等电点为9.07,属于BPI超家族,是稳定的亲水蛋白。亚细胞定位BPI1分布在线粒体(43.5%)、细胞质(30.4%)和细胞核(26.1%)中,表明BPI1可能在嘌呤和嘧啶合成以及能量代谢等过程中发挥信号转导、促进生长等重要作用。二级结构以α-螺旋和β-折叠为主,三级结构呈棒状。同源性及进化树分析表明,实验所得BPI1基因序列与斑点叉尾鲖BPI抗菌肽基因的同源性最高,序列一致性为99.1%,进化树聚为一支,表明BPI1基因编码的蛋白序列在相同物种中的突变率较低,保守性较高,相同物种中其生物活性与个体间的差异关系较小。To research the characteristics and structural features of BPI1 gene from channel catfish（Ictalurus punctatus）,total RNA was extracted from channel catfish kidney tissue and reversely transcribed into c DNA.Specific primers were designed to clone the BPI1 gene. Then BPI1 gene was inserted into the prokaryotic expression vector p TWIN1. The successfully constructed vector p TWIN1-BPI1 was transformed into BL21（DE3）competent cells for prokaryotic expression. According to the analysis of SDS-PAGE,the recombinant expression vector p TWIN1-BPI1 successfully expressed a 50 k Da fusion protein,which conformed to the expected size. BPI1 gene sequence was studied by means of bioinformatics software. The results showed that BPI1 nucleotide sequence encoded 223 aa,with an isoelectric point（p I） of 9.07,and belonged to BPI superfamily. It was a stable and soluble protein. Subcellular localization of BPI1 was in the mitochondria（43.5%）,cytoplasm（30.4%） and nucleus（26.1%）,hence BPI1 may play a role in signal transducer or promote growth,especially in the energy metabolism and cofactor biosynthesis like purines and thymine. The secondary structure of BPI1 mainly consists of α-helices and beta-pleated sheet,besides,the tertiary structure of BPI1 was a rod-like structure. The amino acid sequence was highly conserved and shared the highest homology with BPI of channel catfish,along with sequences identities 99.1% and the same branch in the phylogenetic tree. Thus BPI1 gene sequence had lower mutation rate in the same species and had a strong conservative property that may have little impact on the bioactivity among individuals.

关 键 词：斑点叉尾鲖 BPI1基因 原核表达 生物信息学分析 

分 类 号：Q785[生物学—分子生物学] S941.42[农业科学—水产养殖]