CAJ | 학술논문

按照Promega公司的mRNA提取试剂盒操作手册, 从圆斑蝰蛇(Daboia russellii siamensis)的毒腺中提取mRNA;利用RT-PCR的方法进行体外扩增, 获得C-型凝集素蛋白的基因, 克隆到pMD18-T载体中. 随机挑选14个阳性克隆进行核酸测序, 获得7个编码不同蛇毒C-型凝集素样蛋白亚基的cDNA, 分别命名为DRS-L1、 DRS-L2、 DRS-L3、 DRS-L4、 DRS-L5、 DRS-L6和DRS-L7. 由基因序列推导出的氨基酸序列表明, 克隆到的7个蛇毒C-型凝集素样蛋白的亚基中均有糖识别结构域存在. BLAST分析显示, 仅有DRS-L1的蛋白序列与目前已知的蛇毒C-型凝集素样蛋白的α亚基相似. 序列同源性比较并结合半胱氨酸位点分析, 推测DRS-L1和DRS-L2可能分别是圆斑蝰蛇毒Ⅹ因子激活剂的轻链LC2和LC1. DRS-L3和DRS-L4可能是高分子量的蛇毒C-型凝集素样蛋白的β亚基, 而DRS-L5和DRS-L6可能是低分子量的蛇毒C-型凝集素样蛋白的β亚基. DRS-L7可能是类似于血小板膜糖蛋白Ib结合蛋白的β亚基.
안조Promega공사적mRNA제취시제합조작수책, 종원반호사(Daboia russellii siamensis)적독선중제취mRNA;이용RT-PCR적방법진행체외확증, 획득C-형응집소단백적기인, 극륭도pMD18-T재체중. 수궤도선14개양성극륭진행핵산측서, 획득7개편마불동사독C-형응집소양단백아기적cDNA, 분별명명위DRS-L1、 DRS-L2、 DRS-L3、 DRS-L4、 DRS-L5、 DRS-L6화DRS-L7. 유기인서렬추도출적안기산서렬표명, 극륭도적7개사독C-형응집소양단백적아기중균유당식별결구역존재. BLAST분석현시, 부유DRS-L1적단백서렬여목전이지적사독C-형응집소양단백적α아기상사. 서렬동원성비교병결합반광안산위점분석, 추측DRS-L1화DRS-L2가능분별시원반호사독Ⅹ인자격활제적경련LC2화LC1. DRS-L3화DRS-L4가능시고분자량적사독C-형응집소양단백적β아기, 이DRS-L5화DRS-L6가능시저분자량적사독C-형응집소양단백적β아기. DRS-L7가능시유사우혈소판막당단백Ib결합단백적β아기.
Total mRNA was extracted from a venom gland of snake Daboia russellii siamensis following the manufacturers protocol of the PolyATtract System 1000 kit purchased from Promega Biotech. cDNAs encoding C-type lectins were amplified by RT-PCR and subcloned into a pMD18-T vector. Fourteen positive clones were selected for nucleotide sequencing and seven cDNAs encoding various snake venom C-type lectin-like protein precursors, designated as DRS-L1, DRS-L2, DRS-L3, DRS-L4 DRS-L5, DRS-L6 and DRS-L7, were obtained. Amino acid sequences of these proteins were deduced and each contains a carbohydrate recognition domain. Of all the deduced protein sequences, only DRS-L1 seemed to represent a closer sequence similarity to α subunits of other known snake venom C-type lectin-like proteins using the BLAST program. Homology comparison combined with analysis of cysteine position indicate that DRS-L1 and DRS-L2 are probably the light chain LC2 and LC1 of factor Ⅹ activator from Daboia russellii siamensis venom, respectively. DRS-L3 and DRS-L4 might be the β subunits of higher molecular weight C-type lectin-like proteins while DRS-L5 and DRS-L6 might be β subunits of lower molecular weight C-type lectin-like proteins. DRS-L7 might be the β subunit of a platelet membrane glycoprotein Ib-binding protein similar to echicetin.