中介蝮毒腺C-型凝集素样蛋白基因的cDNA克隆和序列分析  

Cloning of C-type Lectin-like Protein cDNA from the Venom Glands of Gloydius intermedius and Sequence Analysis

作　　者：陈毓[1] 张卫柱 刘玲玲 杨玉娥[1] 杨章民[1] 

机构地区：[1]陕西师范大学生命科学学院,西安710119 [2]西安回天血液制品有限责任公司,西安710075

出　　处：《基因组学与应用生物学》2015年第6期1129-1136,共8页Genomics and Applied Biology

基　　金：国家自然科学基金(30870303);中央高校基本科研业务费专项资金(GK200902029);国家大学生创新训练计划项目(201310718051)共同资助

摘　　要：C-型凝集素样蛋白是一类钙离子依赖型的具有糖结合活性的非酶蛋白,可结合凝血因子及血小板而影响人体的凝血系统稳态,具有抗凝作用。本研究基于中介蝮(Gloydius intermedius)蛇毒C-型凝集素样蛋白α链和β链的部分核苷酸序列,设计了2对PCR引物,通过RT-PCR技术从中介蝮毒腺中克隆出的α和β链(Gi-CTLPα,Gi-CTLPβ)的全长c DNA,克隆入T载体后,对所获克隆进行了测序,并进行了生物信息学分析。结果表明,Gi-CTLPα、Gi-CTLPβ的开放阅读框(ORF)分别为477 bp和462 bp,分别编码158个和153个氨基酸,信号肽分别为21个和23个氨基酸。蛋白质一级结构同源性分析表明,Gi-CTLPα和Gi-CTLPβ之间的序列相似度为29%,Gi-CTLPα和β分别与日本蝮(Gloydius blomhoffi)的Mamushiginα亚基和β亚基的同源性最高(90%和93%),Gi-CTLPα和Mamushigin的α亚基氨基酸序列的差异主要有13个,Gi-CTLPβ和Mamushigin的β亚基氨基酸序列的差异主要有7个。用蛋白功能预测软件SIFT在线分析表明,Gi-CTLPα中第47和136位的氨基酸差异使得Gi-CTLPα与Mamushiginα的功能明显不同,而Gi-CTLPβ和Mamushiginβ亚基的功能没有明显差异。由于Gi-CTLPα和β链会像Mamushigin那样通过二硫键形成异二聚体,这些一级结构的差异会影响其与靶蛋白的结合特性,这一结果为下一步研究Gi-CTLP的功能及利用奠定了理论基础。Snake C-type lectin-like proteins are non-enzymatic proteins which may disturb the blood coagulation homeostasis by binding carbohydrate chains of coagulant factors and blood platelets in a calcium-dependent way,thus have the potential as anticogulant agents. Based on the partial c DNA sequences of α and β of Gloydius intermedius(Gi-CTLP α and β), primers were designed. The full length c DNA sequences of Gi-CTLP α and βwere cloned from the venom glands of Gloydius intermedius by RT-PCR, and subjected to bioinformatics analysis.The results showed that the c DNA of Gi-CTLP α is 628 bp with an open reading frame of 477 bp, encoding a 158amino-acid protein with the 21 N-terminal amino acids as signal, while the c DNA of Gi-CTLP β is 655 bp with an ORF of 462 bp, encoding a 153 amino-acid protein with the 23 N-terminal amino acid as signal. Bioinformatics analysis showed that the sequence identity of Gi-CTLP α and β is 29% and they shared high homology with Mamushigin α and β chain of Gloydius blomhoffi(90% and 93%, respectively). Primary structural comparison of Gi-CTLP α and β with Mamushigin α and β subunit revealed that there were respectively 13 and 7 amino acid differences between the two counterparts. Online analysis with SIFT software indicated that positions 47 and 136 of Gi-CTLP α would make a functional discrepancy whereas the differences between Gi-CTLP β and Mamushiginβ would not cause significant functional divergence. Same as Mamushigin, Gi-CTLP α and β will form heterodimer by disulfide bond, these primary structural difference may thereby affect its binding characteristics to the target proteins. This work may therefore establish a theoretical basis for further investigation of the function and exploitation of Gi-CTLP.

关 键 词：中介蝮 蛇毒C-型凝集素样蛋白 CDNA 克隆 生物信息学 

分 类 号：Q78[生物学—分子生物学]