Stover C K, Vodkin M H, Oaks E V
Department of Rickettsial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
Anal Biochem. 1987 Jun;163(2):398-407. doi: 10.1016/0003-2697(87)90241-7.
A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.
已设计并测试了一种策略，即使用EcoRI转换接头将5'粘性末端限制片段克隆到λgt11表达载体的独特EcoRI位点中。用产生五种不同5'粘性末端的限制酶消化恙虫病东方体基因组DNA，构建了五个λgt11染色体文库。在未对这五个文库进行扩增的情况下，获得了高达10(7)噬菌斑形成单位的重组噬菌体产量。在这五个λgt11表达文库中获得了先前通过蛋白质印迹分析鉴定的所有八种恙虫病东方体多肽抗原的表位编码序列。在检测的39%的重组体中，重组抗原表达依赖于λgt11 lac启动子诱导。该方法通过消除平端连接并简化未连接的EcoRI末端寡核苷酸的去除，显著提高了基因组λgt11文库构建的效率。
