噬菌体展示禽流感病毒抗原变异性基因片段文库的构建

构建T7噬菌体展示禽流感病毒抗原变异性基因片段文库. 首先， 从Gene Bank中查找筛选禽流感病毒抗原变异性基因, 将其截短、 修饰、 简并后得到禽流感病毒抗原变异性基因微阵列. 其次， 将合成的禽流感病毒抗原变异性基因片段文库扩增、 酶切, 链接到双酶切后的T7噬菌体载体基因上, 构成重组噬菌体DNA. 最后， 重组噬菌体DNA经体外包装和扩增, 得到T7噬菌体展示文库, 并进行T7噬菌体展示文库滴度、 重组率和免疫活性测定. 实验结果表明, 从Gene Bank中查找、 筛选、 剪切和修饰共获得96 258条序列构建T7噬菌体展示文库, 原始文库滴度为3.6×107个菌落/mL, 重组率大于90%. 用禽流感病毒H5N1抗体进行捕获, 经聚合酶链式反应(PCR)鉴定, 得到理想目的条带, 证明噬菌体表面展示蛋白具有抗原活性, 可用于禽流感病毒感染患者的快速检测及抗原表位筛选.

Construction of Phage Display of Antigenic VariabilityGene Fragments Library of Bird Flu Virus#br#

We constructed a T7 phage display of antigenicvariability gene fragment library of bird flu virus. Firstly, we searched and scree ned the antigenic variability gene of bird flu virus from Gene Bank, truncated, modified and degenerated the gene to obtain the microarray of antigenic variability gene of bird flu virus. Secondly, the recombinant phages DNA were constructed by amplifying and digesting the synthesized antigenic variability gene frament library of bird flu virus and linking it to the T7 phage vector gene. Finally, T7 phage display library was obtained by packaging andamplification of the recombinant phage DNA in vitro. The titer, recombination rate and immune activity of T7 phase display library were determined.The experimental results show that a total of 96 258 sequences are obtained by searching, screening, cutting and modifying gene bank. T7 phage display library is constructed withoriginal library titer of 3.6×107 colonies/mL. The recombination rateis more than 90%. The bird flu virus H5N1 antibody is used for capture and theideal target band is obtained and identified by polymerase chain reaction (PCR).The display proteins on the surface of phages have antigenic activities, and can be used for rapid d etection of bird flu virus infection patients and screening of antigenic epitopes of antibodies.