蜂王浆主蛋白2在昆虫细胞-杆状病毒系统中的表达与鉴定

doi:10.16431/j.cnki.1671-7236.2021.10.030

摘要/Abstract

摘要： 本研究旨在利用昆虫细胞-杆状病毒表达系统表达蜂王浆主蛋白2（MRJP2），为后续MRJP2功能的深入研究提供材料。根据GenBank中意大利蜜蜂（Apis mellifera L.）MRJP2基因序列，经PCR扩增、克隆至真核表达载体pFastBac1，构建重组杆状病毒质粒MRJP2-Bacmid并转染至Sf9昆虫细胞，以P2代杆状病毒感染Sf9细胞进行诱导表达，利用层析柱和离子交换柱对表达产物进行纯化，并通过SDS-PAGE、Western blotting及四极杆静电场轨道阱高分辨质谱仪（Q Exactive）对目的蛋白进行分析验证。结果显示，本试验成功构建杆状病毒质粒MRJP2-Bacmid，转染至Sf9昆虫细胞并获得表达产物。SDS-PAGE和Western blotting验证结果表明，本研究成功利用昆虫细胞-杆状病毒表达系统表达出大小约为52 ku的MRJP2重组蛋白，且纯度较高；质谱分析结果显示，该重组蛋白匹配到蜜蜂蛋白质数据库中MRJP2的特异性肽段为39个，序列覆盖率为61%，且与MRJP2的匹配得分最高，进一步确认该重组蛋白为MRJP2。本研究利用昆虫细胞-杆状病毒表达系统成功表达出MRJP2，为后续该蛋白生物学功能的深入研究奠定了基础。

关键词: 蜂王浆主蛋白2(MRJP2); 昆虫细胞-杆状病毒表达系统; 蛋白纯化

Abstract: The aim of this study was to obtain major royal jelly protein 2 (MRJP2) by insect cell-baculovirus expression system, so as to get basic materials for subsequent in-depth study of functional research on MRJP2. According to the MRJP2 gene sequence of Apis mellifera L. in GenBank, MRJP2 gene was amplified by PCR and cloned into the eukaryotic expression vector pFastBac1. The recombinant baculovirus MRJP2-Bacmid plasmid was constructed and transfected into Sf9 insect cells, with P2 generation baculovirus infects Sf9 cells to induce expression. The expressed product was purified by chromatography column and ion exchange column, and the target protein was analyzed and identified by SDS-PAGE, Western blotting and Q Exactive. The results showed that this experiment successfully constructed the baculovirus plasmid MRJP2-Bacmid, transfected into Sf9 insect cells and obtained the expression product. The verification results of SDS-PAGE and Western blotting showed that the MRJP2 recombinant protein (52 ku) with high purity was successfully expressed in insect cell-baculovirus expression system. The results of mass spectrometry analysis showed that the recombinant protein matched 39 unique peptides of MRJP2 in the honeybee protein database, with a sequence coverage of 61%, and had the highest matching score with MRJP2. It was further confirmed that the recombinant protein was MRJP2. In this study, the insect cell-baculovirus system was used to successfully express MRJP2, which laid a foundation for the subsequent in-depth study of the biological function of the protein.

Key words: major royal jelly protein 2 (MRJP2); insect cell-baculovirus expression system; protein purification

中图分类号:

S896.3

段香媛, 武江利, 魏俏红, 冯毛. 蜂王浆主蛋白2在昆虫细胞-杆状病毒系统中的表达与鉴定[J]. 中国畜牧兽医, 2021, 48(10): 3779-3786.

DUAN Xiangyuan, WU Jiangli, WEI Qiaohong, FENG Mao. Expression and Identification of Major Royal Jelly Protein 2 in Insect Cell-baculovirus System[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3779-3786.

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