摘要
猪圆环病毒2型(porcine circovirus type 2,PCV2)可读框2(open reading frame 2,ORF2)基因编码具有多个抗原表位的病毒衣壳(Cap)蛋白,能在机体内有效引发免疫反应并产生中和抗体,是预防和治疗猪圆环病毒病(porcine circovirus-associated disease,PCVAD)的主要抗原。但Cap蛋白高昂的生产成本限制了它的广泛应用。为了探索Cap蛋白新的生产途径,本研究选择烟草叶绿体为表达平台,构建了ORF2抗原基因烟草叶绿体表达载体pNK-a03,并通过基因枪法(gene biolistics)将表达载体导入烟草叶绿体中。转化植株经PCR和RT-PCR检测,证实ORF2抗原基因已整合进烟草叶绿体基因组中且正常转录。蛋白质印迹检测和ELISA分析表明,Cap蛋白在烟草叶绿体内获得有效表达,具有免疫活性。此外,种子萌发研究结果显示,转基因烟草植株F1代具有aadA抗性,目的基因可正常进行遗传。这些研究结果为探索Cap蛋白的新型表达途径提供了理论基础。
The porcine circovirus type 2(PCV2)capsid(Cap)protein encoded by the open reading frame 2(ORF2)gene can effectively trigger immune response and produce neutralizing antibodies,which is the key to protect pigs from porcine circovirus-associated disease(PCVAD).However,high production costs limit the widespread use of the Cap protein.To explore a new production pathway of the Cap protein,we chose tobacco chloroplasts as expression platform and constructed the chloroplast expression vector pNK-a03.The expression vector was introduced into tobacco chloroplasts via gene biolistics.Transformed plants were obtained and verified by PCR and RT-PCR,the results showed that the ORF2 antigen gene has been inserted into the tobacco chloroplast genome and normally transcribed.The evaluation of the Cap protein immunogenicity revealed that the crude protein extracts from transgenic plants manifested an immunogenicity against PCV-2 Cap antibody.Most importantly,the seeds from transgenic tobacco can give rise to resistant progeny,indicating that the target gene is hereditary.These results provide a theoretical basis for the development of new production way of the Cap protein.
作者
林优红
程霞英
杨东风
姜永厚
邢少辰
韦正乙
韩蕊莲
梁宗锁
杨宗岐
LIN You-Hong;CHENG Xia-Ying;YANG Dong-Feng;JIANG Yong-Hou;XING Shao-Chen;WEI Zheng-Yi;HAN Rui-Lian;LIANG Zong-Suo;YANG Zong-Qi(Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province,College of Life Science and Medicine,Zhejiang Sci-Tech University,Hangzhou 310018,China;Institute of Agricultural Biotechnology,Jilin Academy of Agricultural Sciences,Changchun 130033,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2020年第3期350-356,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.31570370)
浙江省自然科学基金项目(No.LY15C020005)资助。
