黏虫clip型丝氨酸蛋白酶基因的克隆及原核表达

doi:10.7668/hbnxb.20192510

摘要/Abstract

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2 010 bp,开放阅读框为1 593 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。

关键词: 黏虫, 丝氨酸蛋白酶, 基因克隆, 序列分析, 原核表达

Abstract:

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 2 010 bp,and the open reading frame of 1 593 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.

Key words: Mythimna separate, Serine protease, Gene cloning, Sequence analysis, Prokaryotic expression

连凯琪, 纪爽, 周玲玲, 时志琪, 王飞, 张元臣. 黏虫clip型丝氨酸蛋白酶基因的克隆及原核表达[J]. 华北农学报, 2022, 37(1): 195-201. doi: 10.7668/hbnxb.20192510.

LIAN Kaiqi, JI Shuang, ZHOU Lingling, SHI Zhiqi, WANG Fei, ZHANG Yuanchen. Cloning and Prokaryotic Expression of a clip-type Serine Protease Gene from Mythimna separate[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 195-201. doi: 10.7668/hbnxb.20192510.

http://www.hbnxb.net/CN/Y2022/V37/I1/195

图/表 7

表1 试验所用引物序列

Tab.1 Primer sequences used in the study

引物名称 Primer name	引物序列(5'—3') Primer sequence	引物用途 Primer function
Ser-PRF	AGGACTTTCAGGCGGTGTTCTAT	克隆部分基因
Ser-PRR	AGCGACCATTCTCGTTCACCATTA
5GSP	ATTGCCGTCCAGCGTTGAACAGAG	5'端扩增
5NGSP	TACTCGGTGGCTTAGTTACGCCCG
3GSP	TACCCTACGAAGCCGAGTGTGCCTG	3'端扩增
3NGSP	AGAGAGTCGTGAGACACCGAGGCT
pr1F	GAATTCAGTGACTCCGACGTCAAG	引入酶切位点
pr1R	AAGCTTTTACTGGGCGTTTTTCTGGATCC

图1 MsPG基因的PCR产物
M.DL2000 DNA 标记;1.MsPG片段的PCR产物;2.5' RACE产物;4.3' RACE产物;3,5.5' RACE 和 3' RACE的阴性对照。

Fig.1 PCR products of MsPG gene
M.DL2000 DNA Marker;1.PCR product of partial MsPG gene;2.PCR product of MsPG by 5' RACE;4.PCR product of MsPG by 3' RACE;3,5.The negative control of 5' RACE and 3' RACE.

图2 MsPG基因的核苷酸序列和推导的氨基酸序列
信号肽序列用浅蓝色阴影表示;活性中心(GDSGGP)用红色阴影表示;下划线部分为clip结构域;6个半胱氨酸由方框标记。

Fig.2 Nucleotide and deduced amino acid sequences of MsPG gene
The signal peptide sequence is indicated by light blue shading;the active center(GDSGGP)is indicated by red shading;the underlined part is the clip domain;six cysteines are boxed.

图3 黏虫与其他昆虫间丝氨酸蛋白酶氨基酸序列的多重联配
下划线表示氨基酸序列为活性中心GDSGGP。深色背景表示一致性为100%,红色背景表示一致性大于等于75%,浅蓝色背景表示一致性大于等于50%。HvB5.烟芽夜蛾(登录号:PCG78401.1);HaPE.棉铃虫(登录号:XP_021187378.1);S1TS.斜纹夜蛾(登录号:XP_022821357.1);AtX1.脐橙螟(登录号:XP_013191307.1);BaPS.丛林斜眼蝶(登录号:XP_023945684.1);PxPZ.柑橘凤蝶(登录号:XP_013176912.1);PpPE.玉带凤蝶(登录号:XP_013135138.1);DpPE.黑脉金斑蝶(登录号:XP_032527749.1);GmPE.大蜡螟(登录号:XP_026758758.1);AtX2.脐橙螟(登录号:XP_013191309.1);ApUN.灯蛾属(登录号:CAB3239924.1);MsPE.烟草天蛾(登录号:XP_030031627.1)。

Fig.3 Multiple alignment of amino acid sequences for serine protease between Mythimna separate and other insects
The underlined amino acid sequence GDSGGP is the active center.The dark background indicates that the consistency is 100%,the red background indicates that the consistency is greater than or equal to 75%,and the light blue background indicates that the consistency is greater than or equal to 50%.HvB5.Heliothis virescens(GenBank No.PCG78401.1);HaPE.Helicoverpa armigera(GenBank No.XP_021187378.1);S1TS.Spodoptera litura(GenBank No.XP_022821357.1);AtX1.Amyelois transitella(GenBank No.XP_013191307.1);BaPS.Bicyclus anynana(GenBank No.XP_023945684.1);PxPZ.Papilio xuthus(GenBank No.XP_013176912.1); PpPE.Papilio polytes(GenBank No.XP_013135138.1);DpPE.Danaus plexippus(GenBank No.XP_032527749.1);GmPE.Galleria mellonella(GenBank No.XP_026758758.1);AtX2.Amyelois transitella(GenBank No.XP_013191309.1);ApUN.Arctia plantaginis(GenBank No.CAB3239924.1);MsPE.Manduca sexta(GenBank No.XP_030031627.1).

图4 黏虫和其他物种丝氨酸蛋白酶氨基酸序列的系统进化树

Fig.4 Phylogenetic tree of serine protease among Mythimna separate and other species based on amino acid sequences

图5 去除信号肽的MsPG ORF基因PCR产物和重组质粒双酶切鉴定
M.DL2000 DNA 标记;1,2.去除信号肽的MsPG ORF基因的PCR产物;3.重组质粒pET30a(+)-MsPG双酶切产物。

Fig.5 PCR product of MsPG ORF gene without signal peptide sequence and double digestion identification of the recombinant plasmid
M.DL2000 DNA Marker;1,2.PCR product of MsPG ORF gene without signal peptide sequence;3.Double digestion product of the recombinant plasmid pET30a(+)-MsPG.

图6 诱导表达融合蛋白MsPG的SDS-PAGE分析
M.蛋白质分子质量标准;1—5.分别为pET30a(+)-MsPG在40,35,30,25,20 ℃下的诱导表达;6.pET30a(+)的诱导表达。

Fig.6 SDS-PAGE analysis of fusion protein MsPG expressed by induction
M.Protein Marker;1—5.Induced expression of pET30a(+)-MsPG at 40,35,30,25,20 ℃,respectively;6.Induced expression of pET30a(+).

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