海蚯蚓蛋白酶基因的原核表达及其抗肿瘤活性  

Prokaryotic expression and anti-tumor activity of protease from Arenicola cristata

作　　者：于文静[1] 鞠吉雨[2] 初金鑫[1] 张金宝[2] 连波[1] 杜长青[1] 赵春玲[1] 

机构地区：[1]潍坊医学院药学与生物科学学院,山东潍坊261053 [2]潍坊医学院基础医学院,山东潍坊261053

出　　处：《中国生物制品学杂志》2015年第5期488-492,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(30901779);山东省自然科学基金项目(ZR2009CM019);山东省高等学校科技计划项目(J11LC52)资助

摘　　要：目的原核表达海蚯蚓蛋白酶基因,探讨其对肿瘤细胞增殖的抑制作用。方法根据环节动物门蚯蚓和单环刺螠丝氨酸蛋白酶c DNA序列多重比对获得的保守序列设计引物,从海蚯蚓肠道组织提取总RNA,经3′RACE和5′RACE技术分别克隆海蚯蚓蛋白酶c DNA序列的3′端和5′端,并进行拼接,对其编码的蛋白质作生物信息学分析;将海蚯蚓蛋白酶成熟肽的编码序列亚克隆至原核表达载体p ET-21a(+),转化大肠埃希菌BL21(DE3)进行诱导表达,采用组氨酸亲和层析柱纯化;MTT法检测重组蛋白的抗肿瘤活性。结果经3′端RACE技术获得约250 bp的海蚯蚓蛋白酶基因c DNA 3′端序列,再经5′RACE技术扩增获得约770 bp的c DNA 5′端序列,拼接可得海蚯蚓蛋白酶c DNA序列全长为880 bp,其编码蛋白含270个氨基酸,具有高度保守的GDSGGP序列;重组蛋白相对分子质量为27 000,主要以包涵体形式表达,约占菌体总蛋白的30%,上清纯化后纯度可达95%;重组蛋白酶对人肿瘤细胞增殖具有明显的抑制作用。结论原核表达了海蚯蚓蛋白酶,可明显抑制人肿瘤细胞的增殖,为抗肿瘤基因工程药物的研发提供了实验依据。Objective To express the protease of Arenicola cristata in prokaryotic cells and investigate its inhibitory effect on proliferation of tumor cells. Methods Primers were designed based on the highly conserved sequence obtained by the alignment of protease c DNAs from annelid animals such as Eisenia fetida and Urechis unicinctus. Total RNA was extracted from the intestine tissue of A. cristata, and the 3′-and 5′-terminuses of c DNA sequences were cloned by 3′ RACE and 5′ RACE respectively and spliced, by which the encoded protein was analyzed for bioinformatics. The gene sequence encoding the mature peptide of A. cristata protease was subcloned to prokaryotic expression vector p ET-21a（＋）, and the constructed recombinant plasmid was transformed to E. coli BL21（DE3） and induced with IPTG. The expressed protein was purified by His Band chromatography, and determined for anti-tumor activity by MTT assay. Results The 3′-terminus of c DNA from A. cristata, at a length of about 250 bp, was obtained by 3′ RACE, then the 5′-terminus at a length of about 770 bp by 5′ RACE, with which the c DNA sequence at a full-length of 880 bp was obtained by splicing. The c DNA encoded a protein consisting of 270 amino acid residues and contained highly conserved GDSGGP sequence, which might belonged to serine protease family. The recombinant protein, with a relative molecular mass of 27 000, existed in a form of inclusion body, and contained about 30% of total somatic protein, of which the supernatant reached a purity of 95% after purification. The recombinant protease showed significantly inhibitory effect on the proliferation of human tumor cells.Conclusion The protease from A. cristata was expressed in prokaryotic cells, which inhibited the proliferation of human tumor cells significantly, and provided an experimental basis for development of gene engineering drugs for tumor.

关 键 词：海蚯蚓 丝氨酸蛋白酶 原核细胞 基因表达 抗肿瘤活性 

分 类 号：R730.54[医药卫生—肿瘤]