摘要
为研究蟾蜍皮肤中多肽类有效成分,通过菌落PCR(polymerase chain reaction)对日本蟾蜍(Bufojaponicus formosus)皮肤cDNA质粒文库进行筛选,对测序结果进行分析及同源性比较,获得转胶蛋白-2(transgelin-2,TAGLN2)的5′端缺失的部分cDNA序列(GenBank登录号为JX197456),长为997 bp,包括348 bp的开放阅读框(open reading frame,ORF)、5'端31 bp及3'端618 bp的非翻译区(untranslated region,UTR),编码由115个氨基酸残基组成的蛋白质。推导的氨基酸序列含有一个调宁蛋白同源性结构域(calponin homology,CH)、两个可形成二硫键的半胱氨酸残基、3个α螺旋区域和5个潜在的磷酸化位点。氨基酸序列同源性分析显示,本文获得的日本蟾蜍N-端截短型TAGLN2与热带爪蟾(Xenopus(Silurana)tropicalis)和非洲爪蟾(Xenopus laevis)相对应部分的同源性分别为90%和89%,与其他7种动物的同源性介于71%～85%之间。
To study the bioactive polypeptides included in Bufo skin and its secretions the plasmid skin cDNA library of adult Japanese toad Bufo japonicus formosus was prepared. The pSD64TR has been used as the vector and the cloning sites are Xho I and EcoR I. To screen cDNAs encoding bioactive components, the plasmid cDNA library was transformed into E. coli DH5 competent cells, and positive colonies were screened by colony PCR （polymerase chain reaction）. The suspension of a single colony in LB medium was used as the template, SP6 （the upstream primer of the plasmid cDNA library） and a primer with Xho I site and polyT were used as the primers. As the result, 465 positive colonies out of 1 344 were obtained and their plasmid were collected and sequenced. By homologous analysis, it was found that one of the cDNAs encoding a peptide with high homolog with transgelin-2, which was registered in GenBank （accession number： JX197456）, and it was indicated as a partial cDNA sequence with a deletion at the 5＇ end. The transcript is 997 bp consisting of 31 bp 5＇, 618 bp 3＇ untranslated region （UTR） and an open reading frame （ORF） of 348 bp encoding a polypeptide of 115 amino acids. In the putative protein product, there is a calponin homology domain, two cysteine residues for a disulfide bond and three α-helix domains, and five potential phosphorylation sites. The homologous analysis indicates 90% similarity with Xenopus （Silurana） tropicalis and 89% with Xenopus laevis, and 71%？85% with other species.
出处
《药学学报》
CAS
CSCD
北大核心
2013年第2期250-254,共5页
Acta Pharmaceutica Sinica
基金
国家自然科学基金资助项目(31071181)
2011年度浙江省科技创新活动计划项目(2011R412042)
