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当前位置: 首页 > 文章 > 苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究 应用昆虫学报 2016,53 (4) 706-715
Position: Home > Articles > cDNA cloning and prokaryotic expression of a serine protease from Heliothis viriplaca Chinese Journal of Applied Entomology 2016,53 (4) 706-715

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

作  者：
李莉莉;周晓群;赵奎军;雷蕾;樊东
单  位：
东北农业大学农学院
关键词：
苜蓿夜蛾;丝氨酸蛋白酶;克隆;原核表达;酶活性
摘  要：
【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。
译  名：
cDNA cloning and prokaryotic expression of a serine protease from Heliothis viriplaca
作  者：
LI Li-Li;ZHOU Xiao-Qun;ZHAO Kui-Jun;LEI Lei;FAN Dong;College of Agronomy, Northeast Agricultural University;
关键词：
Heliothis viriplaca;;serine protease;;cloning;;prokaryotic expression;;enzyme activity
摘  要：
[Objectives] Serine protease(SP) is an important proteolytic enzymes, in which serine is an active center. In insects, serine protease is involved in several important physiological processes, such as digestion, growth, the innate immune response and tissue reconstruction. A full-length cDNA sequence of serine protease was obtained from Heliothis viriplaca and its deduced amino acid sequence expressed in an E. coli prokaryotic expression system. Enzyme activity was determined after purification and renaturation. [Methods] Total midgut RNA was isolated from H. viriplaca and one serine protease cDNA sequence was cloned by RT-PCR and RACE to obtain the full-length cDNA sequence. The cDNA sequence was expressed in an E. coli expression system and the expressed fusion protein was denatured, purified and renatured. The activity of the recombinant protein was determined with BTEE as the substrate. [Results] The serine protease cDNA sequence from H. viriplaca was named Hv SP, which was deposited in Gen Bank with the accession number KT907053. The sequence length was 1 017 bp which included an open reading frame of 886 bp, encoding 295 amino acids. The molecular weight was about 30.8 ku, and the isoelectric point was 8.27. The similarity of the Hv SP protein with that of other lepidopteran insects was 46%-92%. The activity of the recombinant protein was 28.7 U/m L at a p H of 8.5 in Tris-HCl buffer solution. The results of quantitative PCR showed that Hv SP was expressed at the m RNA level in multiple tissues, especially in the midgut, but not in the salivary gland. [Conclusion] A new H. viriplaca serine protease cDNA sequence was obtained. The recombinant protein expressed in E. coli was active after denaturation, purification and renaturation. These results establish the basic physiological and biochemical functions of serine protease in H. viriplaca.

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