体外突变新技术:DNA Shuffling技术

体外DNA shuffling技术是一种快速、高效并且实用的分子定向进化技术.和以往的基因突变技术不同,它是一种高通量的突变筛选技术,可以对靶序列进行多次重组和选择,利用该技术建立的突变文库(嵌合文库)具有容量大、多样性好等优点,且更易于实现有益突变的积累.DNA shuffling技术已广泛应用于酶、药物蛋白及其它功能蛋白的改造,并取得了相当大的成功,显示了该技术在蛋白质改造和分子育种方面的潜在应用价值.

应用DNA shuffling及T7噬菌体展示技术定向进化并筛选鉴定sCD74突变体

目的 利用DNA shuffling技术,结合17噬菌体表面展示技术,定向进化MIF受体sCD74.以获得高亲和力突变体基因.方法 定点突变的牛、小鼠、人CD74胞外同源区基因以Dnase Ⅰ消化,回收25-50 bp片段进行DNA shuff-ling;将500 bp shuffling产物经限制性酶切后与T7噬菌体DNA连接,与17 package extracts组建噬菌体文库;采用竞争淘洗方法进行5轮筛选,富集得到高亲和力结合MIF的噬菌体;挑取20株噬菌体单克隆进行基因克隆并测序,对测序结果进行生物信息学分析,同时以竞争ELISA初步鉴定进化效果.结果 20株噬菌体单克隆具有相同的sCD74突变基因.为相同克隆,将此克隆命名为sHCD74m,其基因命名为sHCD74mD;竞争性ELISA分析显示其MIF结合力提高.结论 得到了与MIF高亲和力结合的sCD74突变体基因sHCD74mD.

Background: Diagnostic microbial isolates of bio-safety levels 3 and 4 are difficult to handle in medical field camps under military deployment settings. International transport of such isolates is challenging due to restrictions by the International Air Transport Association. An alternative option might be inactivation and sequencing of the pathogen at the deployment site with subsequent sequence-based revitalization in well-equipped laboratories in the home country for further scientific assessment. Methods: A literature review was written based on a PubMed search. Results: First described for poliovirus in 2002,de novo synthesis of pathogens based on their sequence information has become a well-established procedure in science. Successful syntheses have been demonstrated for both viruses and prokaryotes. However, the technology is not yet available for routine diagnostic purposes. Conclusions: Due to the potential utility of diagnostic sequencing and sequence-basedde novo synthesis of pathogens, it seems worthwhile to establish the technology for diagnostic purposes over the intermediate term. This is particularly true for resource-restricted deployment settings, where safe handling of harmful pathogens cannot always be guaranteed.