扬州大学学报（农业与生命科学版）2024，Vol.45Issue(5)：45-51,7.DOI:10.16872/j.cnki.1671-4652.2024.05.006
凤仙花坏死斑病毒NP基因原核表达及抗血清制备
Prokaryoticexpression and antiserum preparation of NP gene of impatiens necrotic spot virus
摘要
Abstract
Impatiens necrotic spot virus(INSV)is capable of causing serious diseases in a wide range of agricultural and horticultural ornamental crops,and the disease is widespread worldwide.The establishment of a rapid and effective detection method is important for the prevention and control of INSV.In this study,we used a pair of primers based on the sequence of INSV Nuclear Protein to clone the 789 bp gene.Then ligated the target gene to the prokaryotic expression vector pET28a to get the recombinant plasmid pET28a-NPINSV,which was transformed into Escherichia coli Rosetta strain,and then induced by IPTG.The SDS-PAGE showed that the molecular weight of the target gene was about 1 000 mg.After IPTG induction,SDS-PAGE showed a target protein band at a molecular weight of about 28.7 kDa,which was consistent with the expected INSV NP.The INSV NP protein was purified using column affinity and immunised against healthy New Zealand Large White rabbits to prepare rabbit polyclonal antisera.In this study,the INSV NP polyclonal an-tiserum was tested by Dot blot and EL AS A,and the results showed that it was able to specifically recognise the NP protein of INSV.It could be applied to the detection of different environmental samples:the expression of NP protein could be de-tected in peach leaf samples infested with INSV,while it failed to be detected in the leaves of healthy plants.When the prepared antiserum was diluted to 1:20 000 with ultrapure water,INSV NP could still be specifically detected.It indi-cates that the INSV antiserum prepared by E.coli expressing NP is more specific and more potent,and it can provide an effective method reference for the rapid detection of INSV.关键词
凤仙花坏死斑病毒/NP蛋白/原核表达/抗血清Key words
impatiens necrotic spot virus/NP protein/prokaryotic expression/antiserum分类
农业科技引用本文复制引用
蒋润州,纪开燕,赵海婷,丁诗文,秦朗,张坤,赵景奎,贺振..凤仙花坏死斑病毒NP基因原核表达及抗血清制备[J].扬州大学学报（农业与生命科学版）,2024,45(5):45-51,7.基金项目
国家自然科学基金资助项目(32272485) （32272485）
泰州市"凤城英才计划"双创引进专项(2022-12) （2022-12）
江苏省自然科学基金资助项目(BK20211323) （BK20211323）
国家重点研发计划政府间国际科技创新合作重点专项(2022YFE0130900) （2022YFE0130900）
江苏省"科技副总"项目(FZ20221535) （FZ20221535）
扬州大学学科交叉高层次青年人才培育项目(2022-11) （2022-11）
扬州大学高端人才支持计划项目(2022-12) （2022-12）
