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目的:克隆得到人丝氨酸蛋白酶抑制因子Hespintor基因,并应用生物信息学方法进行序列分析。方法:提取HepG2细胞总RNA,RT-PCR扩增Hespintor基因片段,连接至pMD 20-T克隆载体中,转化JM109宿主菌,蓝白斑法筛选阳性克隆菌落,菌落PCR及测序鉴定。利用在线工具软件对Hespintor进行信号肽预测、亚细胞定位、结构域、三级结构、基因染色体定位及组织分布表达分析。结果:人丝氨酸蛋白酶抑制因子Hespintor基因全长285bp,共编码94aa,其中1-23aa编码信号肽,符合亚细胞定位于细胞外的预测;53-93aa编码一个典型的Kazal结构域。Hespintor基因的染色体定位于5号染色体短臂3区3带1亚带(5q33.1);正常组织中仅在睾丸有表达,而肿瘤组织中仅在生殖细胞瘤有表达。结论:Hespintor是一个在机体中静止表达的Ka-zal型丝氨酸蛋白酶抑制因子家族的分泌型新成员。
OBJECTIVE: To clone the human serine protease inhibitor Hespintor gene and analyze the sequence by bioinformatics method. Methods: The total RNA of HepG2 cells was extracted. The Hespintor gene fragment was amplified by RT-PCR and ligated into pMD 20-T cloning vector. The recombinant plasmid was transformed into JM109 host strain. The positive colonies were screened by blue-white staining and identified by colony PCR and sequencing. Hespintor was used to perform signal peptide prediction, subcellular localization, domain, tertiary structure, gene chromosomal location and tissue distribution expression analysis using online tools. Results: The human serine protease inhibitor Hespintor gene was 285bp in length and encoded 94aa. Among them, 1-23aa encoded the signal peptide, which was in line with the prediction of subcellular localization of extracellular domain. 53-93aa encoded a typical Kazal domain. The chromosome of Hespintor gene is located on the short arm of chromosome 5 in zone 3 and subband 1 (5q33.1). The normal tissue is only expressed in the testis and the tumor tissue is only expressed in germ cell tumors. Conclusion: Hespintor is a secreted new member of the Ka-zal serine protease inhibitor family that is expressed in the body at rest.