马铃薯StBIN2蛋白的原核表达、酶活分析及多克隆抗体制备

Prokaryotic Expression, Enzyme Activity Analysis and Antibody Preparation of Potato StBIN2 Protein

摘要

BIN2是一种丝氨酸/苏氨酸蛋白激酶，前期研究发现BIN2在植物应对生物和非生物胁迫方面发挥重要调控功能，但对马铃薯StBIN2蛋白缺乏系统研究。为探究马铃薯StBIN2蛋白的生物学功能，采用PCR扩增的方式获得了马铃薯StBIN2基因，通过同源重组方式将StBIN2基因构建到pCold TF表达载体上，获得StBIN2蛋白，利用激酶活性试剂盒测定StBIN2蛋白的激酶活性，并将纯化的StBIN2蛋白注射新西兰大耳兔从而获得多克隆抗体。StBIN2基因全长1 149 bp，在16 ℃、1.0 mmol/L异丙基硫代半乳糖苷（Isopropyl β-D-Thiogalactoside，IPTG）诱导下表达量最高，StBIN2蛋白的激酶活性在1.09 U/L时制备的多克隆抗体特异性较高，满足Western blotting分析。该研究为进一步揭示马铃薯StBIN2蛋白的生物学功能奠定了基础，有助于深入理解StBIN2基因在马铃薯中的功能机制，为马铃薯的遗传改良和抗逆育种提供理论依据和技术支持。

Abstract

BIN2 is a serine/threonine protein kinase. Previous studies have revealed that BIN2 plays a crucial regulatory role in plant responses to both biotic and abiotic stresses. However, there is a lack of systematic research on the potato StBIN2 protein. To investigate the biological functions of the potato StBIN2 protein, the StBIN2 gene was obtained from potato via PCR amplification. The StBIN2 gene was then constructed into the pCold TF expression vector through homologous recombination to obtain the StBIN2 protein. The kinase activity of the StBIN2 protein was measured using a kinase activity kit. Additionally, purified StBIN2 protein was injected into New Zealand white rabbits to generate polyclonal antibodies. The full-length StBIN2 gene is 1 149 bp. The highest expression level of the StBIN2 protein was achieved under induction with 1.0 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 16 ℃. The polyclonal antibodies prepared when the kinase activity of the StBIN2 protein was 1.09 U/L exhibited high specificity, making them suitable for Western blotting analysis. This study would lay a solid foundation for further elucidating the biological functions of the potato StBIN2 protein, contribute to a deeper understanding of the functional mechanisms of the StBIN2 gene in potato, and provide theoretical support and technical assistance for the genetic improvement and stress-resistant breeding of potato.

关键词

马铃薯 / BIN2蛋白 / 原核表达 / 多克隆抗体

Key words

potato / BIN2 protein / prokaryotic expression / polyclonal antibody

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基金资助

四川省自然基金青年基金项目(2025ZNSFSC1104)

西昌学院2025攀登计划人才引进专项项目(RCZ202506)