摘要
目的克隆多房棘球绦虫(Em)的myophilin基因并对其序列进行分析与预测。方法提取Em原头节总RNA,采用RT-PCR技术扩增myophilin基因,将PCR产物连接入p GM-T载体,转化入DH 5α感受态细胞,挑选阳性克隆送测序。采用生物信息学软件对测序结果进行序列比对分析,同时预测编码蛋白的理化性质、二级结构、三级结构、翻译后修饰位点、结构域和抗原表位。结果扩增出长度为576 bp的cDNA片段,包含完整开放阅读框(ORF),编码190个氨基酸,与已知细粒棘球绦虫的序列同源性达99%。预测结果显示,编码蛋白分子式为C_(935)H_(1519)N_(255)O_(285)S_(11),理论分子质量为212 876,PI为8.66。α-螺旋、β-折叠和无规则卷曲在二级结构中所占的比例分别为51.58%、10.00%和38.42%。含有10个翻译后修饰位点,各1个Calponin结构域和CH结构域,8个B细胞抗原表位和6个T细胞抗原表位。结论成功克隆出Em的myophilin基因并对其进行了有效分析和预测。
Objective Cloning the myophilin gene in Echinococcus muhilocularis, analysis and forecast of its sequence.Methods Total RNA was exacted from the protoscoleces of Em and the myophilin gene was cloned by reverse transcription RCR（RT-PCR）.The PCR products were connected into the pGM-T vectors and then transformed into DH for competent cells.Positive clone was sequenced.Bioinformatics softwares were used to analysis the sequencing results and predict the physical and chemical properties, secondary structure, tertiary structure, post- translated modification sites, domains and antigen epitopes of the encoded protein.Results The 576 bp cDNA fragment was cloned, containing the complete open reading frame （ORF）and encoding 190 amino acids. The homology was up to 99% with the known sequence of Eg.Prediction results showed that the molecular formula of the encoded protein was C935H1519N255O285S11.Its theoretical molecular weight was 212876 and isoelectric point was 8.66.Alpha helix, β-sheet, and random coil accounted for 51.58%, 10.00% and 38.42% in the secondary structure, respectively.There were 10 post-translated modification sites,a calponin domain,a CH domain,8 potentail B cell antigen epitopes and 6 potential T cell antigen epitopes in the myophilin protein. Conclusions The myophilin gene was cloned from Em successfully, and effective analysised and predicted.
作者
何顺伟
张耀刚
李超群
彭源
魏晓星
HE Shun-wei ZHANG Yao-gang LI Chao-qun PENG Yuan WEI Xiao-xing(Department of Ecologically-environment Engineering, Qinghai University Department of Medical College, Qinghai University, Qinghai 810016, China)
出处
《青海医学院学报》
CAS
2016年第4期222-227,共6页
Journal of Qinghai Medical College
基金
青海省科技厅项目(N0.2016-ZJ-746)
关键词
多房棘球蚴
亲肌肉抗原
基因
克隆
序列
分析
Echinococcus multilocularis
Myophilin
Gene
Cloning
Sequence
Analysis
