黏虫clip型丝氨酸蛋白酶基因的克隆及原核表达  

Cloning and Prokaryotic Expression of a clip-type Serine Protease Gene from Mythimna separate

作　　者：连凯琪 纪爽 周玲玲 时志琪 王飞 张元臣 LIAN Kaiqi;JI Shuang;ZHOU Lingling;SHI Zhiqi;WANG Fei;ZHANG Yuanchen(School of Biotechnology and Food Science,Anyang Institute of Technology,Anyang 455000,China;Taihang Mountain Forest Pests Observation and Research Station of Henan Province,Linzhou 456550,China)

机构地区：[1]安阳工学院生物与食品工程学院,河南安阳455000 [2]河南省太行山林业有害生物野外科学观测研究站,河南林州456550

出　　处：《华北农学报》2022年第1期195-201,共7页Acta Agriculturae Boreali-Sinica

基　　金：河南省科技攻关计划项目(212102110090);安阳市科技攻关计划项目(2020-47);安阳工学院博士后启动基金(BHJ2020005,BHJ2020006)。

摘　　要：为了克隆黏虫丝氨酸蛋白酶的全基因序列,并进行原核表达,以黏虫为研究对象,利用RT-PCR扩增丝氨酸蛋白酶的部分基因,进而通过RACE技术,得到丝氨酸蛋白酶全基因,通过Blast等软件对获得基因序列和推导的蛋白质序列进行分析,并利用大肠杆菌表达系统对其进行表达。结果表明,成功克隆黏虫丝氨酸蛋白酶全基因,将其命名为MsPG,序列全长2010 bp,开放阅读框为1593 bp,编码530个氨基酸,蛋白质分子质量为67.6 ku,等电点为7.63,且具有含6个半胱氨酸的clip结构域,推测其为clip型丝氨酸蛋白酶。MsPG氨基酸序列与其他鳞翅目昆虫序列一致性在70.00%~91.92%,其中与烟芽夜蛾(GenBank登录号:PCG78401.1)序列一致性最高,达到91.92%。成功构建重组质粒pET30a(+)-MsPG,其在大肠杆菌原核表达系统中0.1 mmol/L IPTG诱导下的最佳表达温度是25℃。综上,黏虫丝氨酸蛋白酶MsPG具有丝氨酸蛋白酶家族保守的功能位点,且能够在体外进行原核表达。To clone the full gene sequence of Mythimna separate serine protease,and express it in vitro,Mythimna separata was used as the research object,the partial gene of serine protease was amplified by RT-PCR,and further the whole gene was obtained by RACE technology.The obtained gene sequence and deduced protein sequence of serine protease were analyzed using Blast software and other softwares.The gene of serine protease was expressed using the Escherichia coli expression system.The results showed that the full serine protease gene from Mythimna separate was successfully cloned and named as MsPG,with the sequence length of 2010 bp,and the open reading frame of 1593 bp,encoding 530 amino acids.Its protein had the molecular weight of 67.6 ku,and the isoelectrical point of 7.63,and owned a clip-type domain containing 6 cysteine,which was speculated as a clip-type serine protease.The amino acid sequence of MsPG had identities of 70.00%-91.92%to those of other Lepidoptera insects,and showed the highest identity(91.92%)to that of Heliothis virescens(GenBank No.PCG78401.1).The recombinant plasmid pET30a(+)-MsPG was successfully constructed and expressed by 0.1 mmol/L IPTG induction in E.coli prokaryotic expression system,with the highest expression at 25℃.In conclusion,MsPG has conserved functional sites in the serine protease family and can be prokaryotically expressed in vitro.

关 键 词：黏虫 丝氨酸蛋白酶 基因克隆 序列分析 原核表达 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]