泡状棘球蚴骨桥蛋白基因克隆及其生物学分析

Cloning and biological analysis of osteopontin from echinococcus multilocularis

目的骨桥蛋白(OPN)在肿瘤的迁徙和转移中发挥重要作用,但目前泡状棘球蚴在宿主体内迁徙和转移机制尚不明确。文中旨在扩增多房棘球绦虫OPN全长基因序列并进行其生物学分析。方法提取泡球蚴原头节总RNA,逆转录成cDNA,从基因数据库中通过BLAST找到可能的OPN基因保守序列片段设计引物,PCR扩增目的基因测序并鉴定其为同源性片段,然后应用RACE方法对该目的基因进行扩增。对得到的泡球蚴OPN全长基因序列进行功能和结构分析。结果泡球蚴OPN cDNA全长1390 bp。相对分子质量为50 363.75,理论等电点(pl)为11.18,其中含有411个腺嘌呤、297个胞嘧啶、334个鸟嘌呤、348个胸腺嘧啶。ORF长度为849 bp,编码641个氨基酸,始于96 bp,止于944 bp。二级结构预测α-螺旋(h)占20.81%,β-折叠(e)占15.26%,β-转角(t)占10.08%,无规则卷曲(c)占53.85%。结论成功克隆了泡球蚴OPN基因并研究分析其序列及蛋白,为其功能研究奠定了基础。

Objective Osteopontin(OPN) plays an important role in the migration and metastasis of tumors, but the mechanism of the migration and metastasis of alveolar echinococcus in the host remains unclear. Methods The total RNA of protocephalum was extracted and reversely transcribed into cDNA. The potential conserved sequence fragments of OPN gene were found from the gene database through BLAST and designed primers. The target gene was amplified by PCR sequencing and identified as homologous fragments and then was amplified by RACE method. The function and structure of the full length OPN gene sequence were analyzed. Results The full length of OPN cDNA is 1390 bp.The relative molecular weight was 50363.75 and the theoretical isoelectric point(PL) was 11.18, which contained 411 adenines, 297 cytosines, 334 guanines and 348 thymines.ORF is 849 bp in length and encodes 641 amino acids, starting at 96 bp and ending at 944 bp.The α-helix(h), β-pleated sheet(e), β-turn(t) and random coil(c) accounted for 20.81%, 15.26%, 10.08% and 53.85% respectively. Conclusion The OPN gene was cloned successfully and its sequence and protein were analyzed, which laid a foundation for its function research.