CAJ | 학술논문

采用Clontech链转换建库试剂盒,建立了中国长白山乌苏里蝮蛇毒腺cDNA文库,从中克隆了金属蛋白酶/解整合蛋白Ussurin,并进行了序列分析.结果显示,Ussurin开框读码序列由1434bp组成,编码478个氨基酸.由核苷酸顺序推导的氨基酸序列可以看出,Ussurin最初的翻译产物是酶原前体;依次含有18氨基酸组成的信号肽,171氨基酸组成的酶原区和由289氨基酸组成的Ussurin(200氨基酸组成的金属蛋白酶结构域、16氨基酸组成的间隔区和73氨基酸组成的解整合蛋白结构域).Ussurin的金属蛋白酶结构域含有3对二硫键;解整合蛋白结构域含有6对二硫键和特征性RGD(精氨酸-甘氨酸-天冬氨酸)结构.其基因序列和结构域组成与GenBank中蛇毒金属蛋白酶/解整合蛋白呈现高度同源性属于P-Ⅱ.氨基酸序列blast比对发现,酶原区和解整链蛋白结构域呈现极高的同源性,而金属蛋白酶结构域却出现了极高的变异,推测这些变异结构区是为了适应不同的底物、不同受体或同一受体的不同结构域.
채용Clontech련전환건고시제합,건립료중국장백산오소리복사독선cDNA문고,종중극륭료금속단백매/해정합단백Ussurin,병진행료서렬분석.결과현시,Ussurin개광독마서렬유1434bp조성,편마478개안기산.유핵감산순서추도적안기산서렬가이간출,Ussurin최초적번역산물시매원전체;의차함유18안기산조성적신호태,171안기산조성적매원구화유289안기산조성적Ussurin(200안기산조성적금속단백매결구역、16안기산조성적간격구화73안기산조성적해정합단백결구역).Ussurin적금속단백매결구역함유3대이류건;해정합단백결구역함유6대이류건화특정성RGD(정안산-감안산-천동안산)결구.기기인서렬화결구역조성여GenBank중사독금속단백매/해정합단백정현고도동원성속우P-Ⅱ.안기산서렬blast비대발현,매원구화해정련단백결구역정현겁고적동원성,이금속단백매결구역각출현료겁고적변이,추측저사변이결구구시위료괄응불동적저물、불동수체혹동일수체적불동결구역.
The metalloproteinases/disintegrins in the snake venom act as platelet aggregation inhibitor by an antagonism against integrin on platelet through its RGD sequence and may play other important role in cell-cell fusion, cell matrix interaction and other cellular function. Ussurin is a new metalloproteinase/disintegrin that was cloned from Gloydius ussuriensis. Poly (A+) RNA was purified from the total RNA preparation from venom gland of a single G. ussuriensis using the poly (A+) tract-mRNA isolation system. A cDNA library was constructed with the SMART PCR cDNA library construction kit. The cDNA library was screened and the positive clones were selected. The full-length cDNA of Ussurin was obtained. The cDNA encoding the Ussurin precursor has a 51bp 5′-UTR, the open reading frame of Ussurin and a 490 bp 3′-UTR, the open reading frame of Ussurin cDNA nucleotide sequence is 1434bp and codes for 478 amino acids with a predicted molecular mass of 53.2kD and an isoelectric point of 5.37. There is no potential N-glycosylation site in the deduced sequence region. Its deduced amino acid sequence consists of four region, a signal sequence of 18 amino acid residues, a zymogen pro-peptide of 171 amino acid residues with a cysteine switch motif (PKMCGVT) in it, a central metalloproteinase domain of 201 amino acid residues containing a conserved zinc-chelating sequence (HEXXHXXGXXH) and a methionine-turn CIM involving zinc banding also, a space sequence between metalloproteinase domain and disintegrin domain of 15 amino acid residues with a conserved T392, T397,S400, which is specific residues of the P-Ⅱ snake venom metalloproteinases, a disintegrin domain of 73 amino acid residues with a characteristic RGD region and six-disulfide bonds. Ussurin belongs to P-Ⅱ class. The cDNA sequence and deduced amino acid sequence of Ussurin precursor were compared with homologous sequence in the GenBank database, the result reveals high degree of homology in sequence and organization pattern of domain with metalloproteinase/disintegrin gene family of other snake species.Compared with the alignment of amino acid sequence of metalloproteinase/disintegrin member, hypervariable regions of this member were revealed, besides they share higher homologous in the zymogen domain. It suggests that the hypervariable regions are the counterparts directly suitable for interacting with different domain of receptors, different receptors or substrates.