[1]王利华,张玉,郑州廷,等.灰飞虱脂肪酶LsLPS的原核表达及Ni-NTA纯化[J].江苏农业学报,2022,38(03):611-616.[doi:doi:10.3969/j.issn.1000-4440.2022.03.005]
　WANG Li-hua,ZHANG Yu,ZHENG Zhou-ting,et al.Prokaryotic expression and Ni-NTA purification of lipase LsLPS from Laodelphax striatellus[J].,2022,38(03):611-616.[doi:doi:10.3969/j.issn.1000-4440.2022.03.005]
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灰飞虱脂肪酶LsLPS的原核表达及Ni-NTA纯化()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
38
期数:
2022年03期
页码:
611-616
栏目:
植物保护
出版日期:
2022-06-30

文章信息/Info

Title:
Prokaryotic expression and Ni-NTA purification of lipase LsLPS from Laodelphax striatellus
作者:
王利华1; 2; 张玉1; 2; 郑州廷2; 曹越2; 纪锐2; 方继朝2
（1.南京农业大学植物保护学院，江苏南京210095；2.江苏省农业科学院植物保护研究所，江苏南京210014）
Author(s):
WANG Li-hua1; 2; ZHANG Yu1; 2; ZHENG Zhou-ting2; CAO Yue2; JI Rui2; FANG Ji-chao2
（1.College of Plant Protection, Nanjing Agricultural University， Nanjing 210095, China；2.Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China）
关键词:
灰飞虱; 脂肪酶; LsLPS基因; 原核表达; Ni-NTA纯化
Keywords:
Laodelphax striatellus; lipase; LsLPS gene; prokaryotic expression; Ni-NTA
分类号:
S435.112+.3
DOI:
doi:10.3969/j.issn.1000-4440.2022.03.005
文献标志码:
A
摘要:
灰飞虱是危害中国水稻的3种主要稻飞虱之一。脂肪酶是脂肪代谢的关键酶，在昆虫发育、繁殖等生理活动中具有重要作用。为了深入研究脂肪酶(LsLPS)在灰飞虱生长发育中的功能，本研究克隆了LsLPS基因ORF序列，并进行了原核表达和His标签融合蛋白的纯化。结果显示，灰飞虱LsLPS开放阅读框长1 293 bp，可翻译成430个氨基酸，含有信号肽序列和PF00151结构域。LsLPS连接到原核表达载体pET28a(+)-SUMO、pCold I后在表达菌株BL21（DE3）中主要以包涵体形式表达，连接到pET43.1a(+)后可在表达菌株BL21(DE3)中可溶性表达。采用pH 8.0、含20 mmol/L咪唑的磷酸缓冲液作为平衡液，Ni-NTA纯化LsLPS-pET43.1a(+)融合蛋白的纯度和产量相对较高。
Abstract:
Small brown planthopper (SBPH), Laodelphax striatellus, is one of the three main rice planthoppers damaging rice in China. Lipase is the key enzyme in fat metabolism and plays an important role in insect development, reproduction and other physiological activities. In order to investigate the function of lipase in the growth and development of SBPH, the ORF sequence of LsLPS gene was cloned, and the fusion protein with a His-tag was purified. The results showed that the open reading frame of LsLPS was 1 293 bp in length and could be translated into 430 amino acids, containing signal peptide sequence and PF00151 domain. LsLPS fusion proteins expressed using prokaryotic expression vectors pET28a(+)-SUMO and pCold I were mainly in the form of inclusion bodies in the expression strain BL21(DE3). After connecting LsLPS with pET43.1a(+) vector, the obtained proteins were expressed in soluble forms in BL21(DE3). The purity and yield of the fusion protein purified by Ni-NTA were relatively higher using phosphate buffer (pH 8.0) containing 20 mmol/L imidazole as equilibrium solution.

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备注/Memo

备注/Memo:
收稿日期：2021-11-30基金项目：江苏省农业科技自主创新基金项目[CX(20)1004]；国家自然科学基金项目（31572004）；江苏省自然科学基金项目（BK20170072）作者简介：王利华（1979-），女，四川仁寿人，博士，研究员，主要从事水稻害虫发生规律及防治技术研究。张玉为共同第一作者。通讯作者：方继朝，（E-mail） fangjc126@126.com；纪锐，（E-mail）411526774@qq.com
更新日期/Last Update: 2022-07-07