管氏肿腿蜂毒液抗凝血酶Ⅲ基因的克隆与表达分析
Cloning and expression of the venom antithrombin Ⅲ gene in Scleroderma guani (Hymenoptera: Bethylidae)
韩开健 樊晓红 吴朝妍 朱家颖
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DOI：10.7679/j.issn.2095-1353.2021.107
作者单位：西南林业大学云南省森林灾害预警与重点实验室，昆明 650224； 西南林业大学西南山地资源保育与利用教育部重点实验室，昆明 650224
中文关键词：管氏肿腿蜂；抗凝血酶Ⅲ；基因克隆；表达特征；原核表达
英文关键词：Scleroderma guani; antithrombin Ⅲ; gene cloning; expression profile; prokaryotic expression
中文摘要：
【目的】 克隆和表达管氏肿腿蜂Scleroderma guani毒液抗凝血酶Ⅲ（Antithrombin Ⅲ）基因SgAT-Ⅲ，为深入研究该毒液基因的生理功能奠定基础。【方法】 利用逆转录PCR技术克隆SgAT-Ⅲ基因开放阅读框（Open reading frame，ORF）序列，使用生物信息学软件分析其基因序列结构特征，通过qPCR技术分析其在雌成虫不同组织中的表达模式，采用载体pCzn1对其进行原核表达。【结果】 克隆得到SgAT-Ⅲ基因的ORF，长1 338 bp，编码446个氨基酸，其中第1-19位氨基端为信号肽，理论分子量49.49 ku，等电点6.23。多序列比对分析表明，SgAT-Ⅲ与蚂蚁AT-Ⅲ具有较高的氨基酸一致性（>64%），C末端具有serpin蛋白家族典型反应中心环区，含有供靶标蛋白识别的活性裂解位点。系统发育分析表明，SgAT-Ⅲ与膜翅目其他昆虫的AT-Ⅲ亲缘关系较近，且与蚂蚁的AT-Ⅲ聚为一支。qPCR分析表明，SgAT-Ⅲ基因在毒液器官中高表达。SDS-PAGE电泳检测发现，成功表达SgAT-Ⅲ重组蛋白，纯化得到高纯度的重组蛋白。【结论】克隆得到SgAT-Ⅲ基因，其在毒液器官中高表达，纯化得到SgAT-Ⅲ重组蛋白，为进一步研究该毒液基因的生理功能奠定了基础。
英文摘要：
[Objectives] The aim
of this study was to clone and express the venom antithrombin Ⅲ gene of Scleroderma
guani (SgAT-Ⅲ) and thereby provide a basis for characterizing the
physiological function of this gene. [Methods] The open reading frame (ORF) sequence of the SgAT-Ⅲ gene was cloned by reverse transcription PCR technology and its
sequence structure analyzed with bioinformatic software. The gene expression
profiles of SgAT-Ⅲ in different developmental stages and tissues of female
adults were detected with qPCR. The gene was expressed using the prokaryotic
vector pCzn1. [Results] The ORF
of the SgAT-Ⅲ gene was successfully cloned and found to be 1 338 bp in
length, encoding 446 amino acids with a signal peptide comprised of amino acids
1-20, a predicted molecular weight of 49.49 ku and an isoelectric point (pI) of
6.23. The results of multiple sequence alignment revealed that SgAT-Ⅲ shares
high amino acid identity (>64%) with SgAT-Ⅲs of other members of the
Formicidae. Its C-terminal sequence has a typical, reactive, center loop region
of the serpin protein family, and contains an active cleavage site that can be
recognized by target protease. Phylogenetic analysis indicates that SgAT-Ⅲ is
closely related to AT-Ⅲs of other hymenopteran insects, particularly those of
ants. qPCR analysis revealed that the SgAT-Ⅲ gene was abundantly
expressed in the venom apparatus. SDS-PAGE electrophoresis detection revealed
that the recombinant SgAT-Ⅲ was successfully expressed, and that high-purity
recombinant SgAT-Ⅲ was obtained. [Conclusion] The SgAT-Ⅲ gene was successfully cloned and
found to be abundantly expressed in the venom apparatus. Obtaining this
purified, recombinant protein lays a foundation for further investigation of
the physiological function of SgAT-Ⅲ.