摘要
孵化酶(hatchingenzyme,HE)是后生动物卵孵化过程中起关键作用的一类蛋白酶。在已完成全长967bp的家蚕孵化酶样基因(BmHEL)克隆及其转录特性分析的基础上,研究了该基因在原核表达系统的表达、产物纯化及其对家蚕卵壳的降解功能。以全长BmHEL质粒DNA为模板,用PCR方法分别获得了该基因的开放阅读框(BmHELORF,885bp)、缺失信号肽编码区(BmHELa,834bp)和推测成熟酶功能编码区(BmHELb,624bp)的3个片段,并亚克隆入原核表达载体pET28a(+),在大肠杆菌(E.coli)BL21中进行表达。表达的3种外源蛋白均以包涵体形式存在,获得含有6×His-Tag标签的融合蛋白,其分子质量分别为33.4、31.8、24.0kD。通过Westernblot方法检测到了用Ni-NTA亲和层析柱纯化的目的蛋白。纯化复性后的3种孵化酶对家蚕卵壳底物有一定降解活性,其中BmHELORF的活性最高,BmHELb的活性最低。在pH7.1反应条件下3种酶的活性比pH9.5时高。研究结果在蛋白质水平上进一步证实家蚕孵化酶样蛋白参与了蚕卵孵化这一重要生命过程。
Hatching enzyme is a kind of protease, which plays a key role in the hatching process of metazoans. We previously reported the cloning and transcriptional characterization of a full length cDNA of hatching enzyme-like gene in Bombyx mori （BmHEL, 967 bp）. Here, we investigated the expression and purification of BmHEL in a prokaryotic expression system and its degradation activity to Bombyx mori eggshells. Using a recombinant plasmid DNA harboring BmHEL as the template, three DNA fragments were obtained by PCR method, including the intact ORF （BmHEL, 885 bp）, one with a deleted signal peptide （ BmHELa, 834 bp） and the predicted mature enzyme （ BmHELb, 624 bp）. Then they were subcloned into expression plasmid pET28a（＋）, respectively. The recombinant plasmids were transferred into E. coli strain BL21 and the target proteins were expressed, which were all present in the cell as inclusion bodies. According to SDS-PAGE results, the molecular weights of the target proteins, which were fused with 6 His-tag, were 33.4 kD, 31.8 kD and 24.0 kD, respectively. They were purified on Ni-NTA affinity columns and identified by Western-blot experiments. All of the purified proteins showed degradation activity against Bombyx moil eggshells. Among them, BmHEL had the highest activity while BmHELb had the lowest. Their activity was higher at pH 7. 1 than at pH 9.5. These results further verified the involvement of BmHEL in the hatching process of Bombyx mori eggs at the protein level.
出处
《蚕业科学》
CAS
CSCD
北大核心
2010年第3期407-412,共6页
ACTA SERICOLOGICA SINICA
基金
国家重点基础研究发展计划“973”项目(No.2005CB-121000)
国家高技术研究发展计划“863”项目(No.2007AA100504)
江苏省高校自然科学基金项目(No.07KJD230052)
关键词
家蚕
孵化酶样基因
原核表达
蛋白纯化
卵壳底物
降解
Bombyx mori
Hatching enzyme-like gene
Prokaryotic expression
Protein purification
Eggshell substrate
Degradation
