生物技术2024,Vol.34Issue(4) :407-411,438.DOI:10.16519/j.cnki.1004-311x.2024.04.0060

人SNARE复合物相关蛋白的表达与纯化

Expression and purification of human SNARE complex associated proteins

支旭勃 陈款民 王昭维 李倩楠 孙雪嫄 张芷萌 杨影 李元
生物技术2024,Vol.34Issue(4) :407-411,438.DOI:10.16519/j.cnki.1004-311x.2024.04.0060

人SNARE复合物相关蛋白的表达与纯化

Expression and purification of human SNARE complex associated proteins

支旭勃 1陈款民 1王昭维 1李倩楠 1孙雪嫄 1张芷萌 1杨影 1李元1
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作者信息

  • 1. 陕西慧康生物科技有限责任公司,陕西西安 710065
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摘要

[目的]构建人SNARE复合物中的SNAP25、Syntaxin 1a和VAMP1蛋白真核表达载体,并于体外进行表达纯化.[方法]将重组质粒转化至大肠杆菌DH10Bac感受态细胞,经抗性和蓝白斑筛选,获得重组杆粒;用脂质体转化法,将重组杆粒转染至Sf9昆虫细胞中使其产生重组病毒,最后对病毒进行扩增后再感染Sf9昆虫细胞表达目的蛋白;所得蛋白进行Western Blot鉴定及纯化.[结果]测序结果表明SNAP25、Syntaxin 1a和VAMP1真核表达载体构建成功,Western Blot检测出三种蛋白的特异性条带,利用His标签蛋白纯化出Syntaxin 1a蛋白.[结论]利用杆状病毒表达系统成功表达出了 SNARE复合物相关蛋白.

Abstract

[Objective]The eukaryotic expression vectors of SNAP25,VAMP1 and Syntaxin 1a protein in human SNARE com-plex were constructed,and then expressed and purified in vitro.[Method]The recombinant plasmid was transformed into com-petent E.coli DH10Bac cells to construct the recombined bacmid and then transfected into Sf9 insect cells by liposome transfor-mation to produce recombinant virus,after that,the virus was amplified and infected Sf9 insect cells to express the target pro-tein.The obtained proteins were identified and purified by Western Blot.[Result]The sequencing results showed that the eu-karyotic expression vectors SNAP25,Syntaxin 1a and VAMP1 were successfully constructed.The specific bands of the three pro-teins were detected by Western Blot,and Syntaxin 1a was purified by His tag protein.[Conclusion]SNARE complex related proteins were successfully expressed by baculovirus expression system.

关键词

SNARE复合物/SNAP25蛋白/Syntaxin/1a蛋白/VAMP1蛋白/重组杆粒/杆状病毒/Sf9昆虫细胞/蛋白纯化

Key words

SNARE complex/SNAP25 protein/Syntaxin 1a protein/VAMP1 protein/recombinant bacmid/baculovirus/Sf9 insect cells/protein purification

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基金项目

西安市博士后创新基地项目(市人社:2019-683号)

出版年

2024
生物技术
黑龙江省微生物学会 黑龙江省生物工程学会 黑龙江省科学院微生物研究所

生物技术

CSTPCD
影响因子:0.611
ISSN:1004-311X
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