利用噬菌体展示技术筛选与RSV病毒P20蛋白结合的单链抗体和短肽-遗传学专业论文.docxVIP

摘要水稻条nt-十?q病毒是一类多组份植物RNA病毒，隶属柔丝病毒组，通过灰飞 摘要 水稻条nt-十?q病毒是一类多组份植物RNA病毒，隶属柔丝病毒组，通过灰飞 虱以持久方式经卵传播。它广泛分布于我国、朝鲜、兰竺蓼孽东部各国’是{撕i 条叶枯病的病原，在流行年份会造成水稻严重减产。IRsV基因组包括4条RNA ／在分离纯化了GST-P20融合蛋白后，以该蛋白为抗原，成功免疫了小鼠。 桶和扶飞虱t1}P20蛋白的存在，而未感染的水稻和灰飞虱则无此蛋白。 通过噬菌体艘示技术，在随机7肽库和12肽库中，经过3轮生物筛选，分 }；Jj筛选到了6个和9个与P20蛋白有结合作用的短肽。筛选到的9个12肽中1 。j短肽和3号短8k有相似的氨基酸序列WxWxxxxW(其中x代表任意的氨基酸)， 得到表达。运用展示短肽的噬菌体，可以检测水稻中RSV病毒的存在。圆二色 得到表达。运用展示短肽的噬菌体，可以检测水稻中RSV病毒的存在。圆二色 光谱表明，短肽与P20结合后，造成了P20的构象变化，推测该系列短肽可以改 变或破坏P20蛋白的功能，可作为抑制病毒复制和传播的候选多肽。同时，根据 筛选的短肽序列，采用了生物信息学方法，尝试分析了P20可能结合和相互作用 的蛋白，推测了P20的功能可能是调节反转录因子的pol多聚蛋白，参与RSV 的复制。 针对RSV的P20蛋白的单链抗体和短肽的筛选，为通过转基因防治水稻条 叶枯病的防治和检测水稻及灰飞虱中RSV病毒打下了坚实的基础；同时，抗P20 单链抗体的克隆和表达，可以作为分析P20蛋白功能和研究RSV病毒同水稻和 灰飞虱相互作用的工具。j一。一一∥。7 凇；、：。越F；?蔓多毒、，岛∽1f咖 ’二o 0一p IIl AbstractRice Abstract Rice s仃ipe disease is caused by Rice Stripe Virus(RSV)，which is transmitted from rice to rice by planthopper(Laodelphax striallus)，and may bring great loss to rice yield．RSV is an RNA virus containing four RNA segments and it belongs to genus Tenuivirus．The viral segment of RNA 4 encodes a 20 kilo Dalton protein(P20 protein)．The P20 protein can be detected in infected rice plant and planthopper except RSV particles shows that P20 protein is not the structural protein of the virus．All these indicated that P20 protein plays aIl important role of virus transmission or replication．In this study，we have selected single chain fragment variable(ScFv)from constructed immunized phage-display library and peptides against P20 protein from phage—displayed random peptide library． A phage display library of ScFv was constructed from spleen of immunized mouse with GST-P20 fusion protein．Several recombinants were obtained after 4 rounds of biopanning The results of enzyme linked immunosorbent assay(ELISA) and Western blot showed that the displayed proteins of these isolates were able to specifically bind the RSV P20 protein．The sequence of the gene had the typical character of variable region of heavy and light chains of antibody．The ninth