噬菌体展示技术和其应用ppt课件.pptVIP

; 噬菌体展示技术是将各种多肽或蛋白质以融合蛋白的形式表达并展示在噬菌体表面，同时将其遗传密码包含于噬菌体内部，这使得蛋白质的功能与其基因密码有机的连结在一起。 ;非展示系统 展示系统;The expression of exogenous peptides on the surface of filamentous bacteriophage was initially described by Smith in 1985. Since his first study, different molecules such as small peptides and antibodies have been displayed on coat proteins of phage, greatly expanding the applications of the technology. The past decade has seen considerable progress in the techniques and applications of phage libraries. In addition, different screening methods have allowed isolation and characterization of peptides binding to several molecules in vitro, in the context of living cells, in animals and in humans.;- In vivo use of phage libraries. Initially the phage library is injected in the circulation. Next, phage is allowed to circulate for minutes or hours (in this case when internalized phages are to be recovered). Finally, after deep anesthesia, mice are euthanized and the organs are dissected for phage recovery and peptide sequencing.;Phage structure. PIII, pVI, pVII, pVIII and pXIX representphage proteins. Exogenous peptides are expressed or “displayed” usuallyon pIII or pVIII.;三 丝状噬菌体的生物学 形态：长杆状，长度约1 um，管直径约6nm。 基因组：约6400核苷酸大小的单链线状DNA， 结构：●2700个主要蛋白（PVIII）分子螺旋状排列形成长管， ●一头5个拷贝的次要壳蛋白PIII和PVI， ●另一头则由次要壳蛋白PVII和PIX。 生理：●F菌毛结合的序列是PIII蛋白N端的200个氨基酸。 ●一个分裂周期中可生产几百个子代， ● ? 其产量可达到0.3mg/ml。 ;外源蛋白的展示部位;;抗体库技术简单流程; 获得抗体基因; 插入载体;; 表达; 筛选;2018/7/28;噬菌体展示技术的发展简史; 噬菌体定义：是感染细菌、真菌、放线菌或螺旋体等微生物的病毒。 ;丝状噬菌体;T7与M13相比优势明显;噬菌体cDNA展示技术 以改构的噬菌体为载体， cDNA基因片段定向插入 噬菌体外壳蛋白基因区， 使外源蛋白表达并展示 于噬菌体表面， 通过亲和富集法筛选 表达有特异蛋白质的噬菌体。;噬菌体展示系统的类型;噬菌体cDNA展示技术的应用;2018/7/28;问题;展望;应用举例：;一、半合成噬菌体抗体库的构建;材料和方法;;结果;图3.3 重链分段PCR扩增结果 1－3泳道：H135，H2，H4(约290bp) 4－7泳道：CDR3-5, CDR3-8, CDR3-10, CDR3-12(约400bp) M：Marker DL2,000 ;图3.4 重链重叠PCR扩增结果 1－3泳道： CDR3-5+(H