中华大蟾蜍galectin-3 cDNA的克隆、序列分析及原核表达载体的构建  

Cloning and Sequancing of Bufo gargarizans Galectin-3 cDNA and the Construction of the Vector for Its Prokayotic Expression

作　　者：徐跃[1] 宋敏国[1] 杨仙玉[1] 袁进强[1] 

机构地区：[1]浙江农林大学林业与生物技术学院,浙江临安311300

出　　处：《西南大学学报（自然科学版）》2012年第4期46-52,共7页Journal of Southwest University(Natural Science Edition)

基　　金：2006年浙江省留学人员科技活动择优基金资助项目;2011年度浙江省科技创新活动计划(新苗人才计划)基金资助项目(2011R412042);2010年度浙江省大学生科技创新活动计划(新苗人才计划)基金资助项目(2010R412003)

摘　　要：通过PCR扩增出中华大蟾蜍Bufo gargarizans半乳糖凝聚素3(gal-3)cDNA全长开放读码框(ORF)并克隆至pGM-T载体,经DNA测序确定获得全长ORF序列(GenBank登录号为JN993733).序列分析表明中华大蟾蜍gal-3的ORF是由789个碱基组成,编码262个氨基酸残基组成的蛋白质.推导氨基酸序列同源性比较发现,中华大蟾蜍与非洲爪蟾Xenopus lavis同源性最高,为51%,与其他10种动物的同源性在41%～50%之间.SMART分析显示gal-3蛋白在136-259位氨基酸存在保守的半乳糖结合域.Net Phos预测gal-3在Ser-6和Ser-186有潜在的丝氨酸磷酸化位点.gal-3氨基酸序列构建的进化树符合传统动物分类学特点.经菌落PCR、双酶切和DNA测序确认gal-3的ORF正确插入原核表达载体pET-28b,表明原核表达用重组质粒构建成功,为研究其蛋白功能和生物学功能奠定了基础.The galectin-3 （gal-3） ORF of Bufo gargarizans was amplified by PCR and cloned into vector pGM-T. DNA sequencing indicated that the ORF of gal-3 was 789 bp in length, encoding 262 amino acid residues （GenBank NO. JN993733）. Homologous analysis indicated that B. gargarizans had a similarity with Xenopus lavis as high as 51%, and a similarity of 41%-50% with other 10 species. A gal-binding domain was predicted at 136-259 a.a. by SMART. Two potential phosphorylation sites of gal-3 （Ser-6 and Ser-186） were found by Net Phos. The phylogenetic tree of gal-3 amino acid sequences was consistent with morphological taxonomic characters. Colony PCR, double-enzyme digestion and DNA sequencing proved that the gal-30RF was inserted correctly into the prokaryotic expression vector pET-28b, which will be the basis for the further studies on the function and biological activity of gal-3 of B. gargarizans.

关 键 词：中华大蟾蜍 半乳糖凝聚素3 基因克隆 原核表达载体构建 

分 类 号：Q959.53[生物学—动物学]