扩展莫尼茨绦虫β微管蛋白基因的克隆及序列分析

【目的】从扩展莫尼茨绦虫中克隆β微管蛋白基因,进行序列测定和生物信息学分析,为进一步研究该基因的功能奠定基础。【方法】构建扩展莫尼茨绦虫成虫cDNA文库,随机挑取重组阳性克隆进行测序,对部分序列进行引物步移法测序,获得其全长cDNA序列;采用生物信息学等技术对该cDNA序列进行开放阅读框(ORF)的寻找、编码氨基酸的推导、核苷酸和氨基酸同源性比较以及蛋白二级结构的初步预测。【结果】获得了1个扩展莫尼茨绦虫新基因——β微管蛋白基因,全长1 571 bp,编码444个氨基酸,与日本血吸虫的β微管蛋白氨基酸序列具有78.6%的同源性。编码蛋白的理论分子量为49.6 ku,等电点为4.96;1～4位氨基酸MREI为β微管蛋白转录后调控信号,140～146位GGGTGAG存在一个微管蛋白标志信号片段,在99～106位和391～410位存在两个典型的β微管蛋白保守区;亚细胞定位分析其为细胞的骨架蛋白。【结论】获得了扩展莫尼茨绦虫β微管蛋白基因的全长cDNA序列,为该基因功能的实验性鉴定工作奠定基础。

Cloning and Sequence Analysis of β-tubulin from Moniezia expansa

【Objective】 The purpose of this project was to clone β-tubulin gene from Moniezia expansa(M.expansa) cDNA library and then sequence and analyze it from the perspective of bioinformatics in order to provide a foundation for further research.【Method】 A cDNA library was constructed from M.expansa.Clones were selected randomly from the cDNA library and were sequenced by using the method of expression sequence tags(ESTs).Novel genes were acquired by primer-walking.The cDNA sequence encoding M.expansa β-tubulin was analyzed,including searching the ORF,translating the nucleotide to protein sequence,similarity searches and secondary structure predication.【Result】β-tubulin genes,1 571 bp and coding for 444 amino acids was got.The β-tubulin of M.expansa and Schistosoma japonicum were homologous with an identity of 78.6 %.The molecular weight of the protein was predicted to be 49.69 Ku,and the academic pI was 4.96.The MREI domain,a β-tubulin mRNA autoregulation signal,was found at the N-terminus.There was a tubulin signature sequence GGGTGAG at the sites 140-146 of the deduced amino sequence and two conserved amino acid sequence of β-tubulin at the sites 99-106 and 391-410.It is a kind of cytoskeletal protein at subcellular location.【Conclusion】 The full-length cDNA encoding M.expansa β-tubulin was obtained,which laid the foundation for further functional study of this gene.