鸭疫里默氏杆菌基因组文库的构建及免疫原性基因初步筛选  

Construction and immunoscreening of partial genomic library of Riemerella anatipestifer

作　　者：朱德康[1] 程安春[1] 汪铭书[1] 丁轲[1] 

机构地区：[1]四川农业大学动物科技学院禽病防治研究中心,四川雅安625014

出　　处：《中国兽医学报》2007年第6期834-837,共4页Chinese Journal of Veterinary Science

基　　金：教育部重点研究项目(地方01102);四川省重大基础研究项目(03JY029-035-1/04JY029-006-1/05JY029-109/2006J13-048);教育部"新世纪优秀人才支持计划(NCET-04-0906)

摘　　要：以血清1型鸭疫里默氏杆菌（RA）CH-1株为材料提取基因组DNA，采用限制性内切酶Sau3AI进行部分酶切消化，纯化回收1．0～6．5kb片段，用T4DNA连接酶将回收片段与经BamHI酶切并去磷酸化的ZAP Express载体进行连接，用噬菌体包装蛋白包装。经测定包装滴度为5.23×10^6pfu／mL，蓝白斑筛选重组率为96．8％，表明已成功构建血清1型RA CH-1株部分基因组文库。在此基础上以RA抗血清为抗体探针进行免疫筛选，获得3个阳性克隆，经多次重复筛选后再体内删除，得到含有插入片段的质粒并测序。生物信息学分析结果显示，该序列（GenBank登录号：DQ151838）含有1个编码369个氨基酸的完整开放阅读框架，是尚未见报道的RA基因序列，并且存在多个预测的抗原表位。The total genomic DNA of CH-1 strain of Riemerella anatipestifer serotype 1 was partially digested with Sau3A I and the 1.0-6.5 kb DNA fragments were recovered from agarose gel. The DNA was ligated with ZAP express predigested vector by T4 DNA ligase and packaged by Gigapack 111 gold packaging extract. The results showed that the packaging titer was 5.23×10^6 pfu/mL and recombinant rate was 96.8%. It is concluded that the constructed genomic library can be used to screen target clones. The constructed genomic library was screened by using the sera of immunized rabbits. A phagemid was gained with in vivo excision of the positive clone. The phagemid was sequenced and then submitted to GenBank with accession number of DQ151838. The nucleotide sequence was analysized with DNAstar software and BLAST program in GenBank. The results showed that the sequence possess a complete open reading frame of 369 amino acids and many predicted antigen epitopes. It is believed to be a valuable candidate for designing new vaccine.

关 键 词：鸭疫里默氏杆菌 免疫筛选 抗原表位 

分 类 号：S852.61[农业科学—基础兽医学] Q78[农业科学—兽医学]