草地贪夜蛾β-Actin基因的原核表达及多克隆抗体制备  

Prokaryotic Expression and Polyclonal Antibody Preparation of the β-Actin in Spodoptera frugiperda

作　　者：罗海玲 夏顺超 卢琴 李海银 Luo Hailing;Xia Shunchao;Lu Qin;Li Haiyin(Institute of Entomology,Guizhou University/The Provincial Key Laboratory for Agricultural Pest Management of Mountainous Region of Guizhou,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州大学昆虫研究所/贵州省山地农业病虫害重点实验室,贵州贵阳550025

出　　处：《山地农业生物学报》2023年第2期81-85,共5页Journal of Mountain Agriculture and Biology

基　　金：贵州省科技计划项目(黔科合基础-ZK[2021]一般102);贵州大学大学生创新创业训练计划项目(贵大省创字[2021]083);贵州大学“SRT计划”项目(贵大SRT字[2021]252)。

摘　　要：草地贪夜蛾是一种重要的农业害虫,其迁飞性强、寄主广、繁殖力高,给我国农业经济带来了严重损失。从分子水平探究草地贪夜蛾生长、发育、繁殖、抗药性形成的内在分子机理,能为虫害的防治、防控提供重要参考。β-Actin作为一种高度保守的看家蛋白常在分子生物学中用作内参去定量目标蛋白的表达水平,因而获得高质量的β-Actin抗体是从事相关分子研究的基本前提。因此,本研究克隆了草地贪夜蛾β-Actin基因部分编码序列,长度为579 bp。利用原核表达系统诱导获得了约37 kD的β-Actin重组蛋白,将纯化后的重组蛋白免疫新西兰大白兔制备得到β-Actin多克隆抗体血清。经ELISA检测,获得的抗血清效价达1∶256000。利用制备的β-Actin抗血清进行Western blot试验,结果显示在42 kD处出现强且单一的蛋白条带,说明制备的β-Actin抗血清能有效识别草地贪夜蛾β-Actin蛋白。综上,本研究制备的β-Actin多克隆抗体效价高、特异性较强,能为草地贪夜蛾后续相关分子研究提供基本条件。Spodoptera frugiperda is an important agricultural pest.This pest has wide range of hosts, high reproduction, and strong immigration, which cause serious losses to the agricultural economy in China.The exploring of molecular mechanism of the growth, development, reproduction, and drug resistance of S.frugiperda,which could provide important information for the control of S.frugiperda.β-Actin, a highly conservative housekeeper protein, is often used as an internal reference to quantify the target protein expression frequently.Therefore, a high-quality β-Actin antibody is a prerequisite for protein research.In this study, we cloned the partial coding sequences of the β-Actin gene(579 bp) from S.frugiperda.A prokaryotic expression system was used to induce the β-Actin recombinant protein of about 37 kD.And then purified β-Actin recombinant proteins were used to immunize New Zealand white rabbit for preparing the antiserum β-Actin.ELISA analysis showed that the titer of the β-Actin antiserum was 1∶256000.Western blot showed that the β-Actin antibody could specifically recognize β-Actin protein(the band was about 42 kD).In conclusion, the β-Actin polyclonal antibody with high potency and specificity was generated successfully in this study, which will provide the basic conditions for further molecular research in S.frugiperda.

关 键 词：草地贪夜蛾 Β-ACTIN 原核表达 多克隆抗体 

分 类 号：Q965[生物学—昆虫学]