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  

Committed differentiation,screening and verification of an sCD74 mutant by DNA shuffling and T7 phage display

作　　者：臧家涛[1] 李淑慧[1] 李鹏[1] 陈安[1] 胡川闽[1] 

机构地区：[1]第三军医大学医学检验系临床生物化学教研室,重庆400038

出　　处：《第三军医大学学报》2008年第8期694-697,共4页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(2007BB5069);第三军医大学青年研究基金(XG200512)~~

摘　　要：目的利用DNA shuffling技术，结合T7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DNaseⅠ消化，回收25—50bp片段进行DNA shuffling；将500bp shuffling产物经限制性酶切后与T7噬菌体DNA连接，与T7 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。Objective To prepare a mutant DNA of sCD74, the receptor of MIF, by the technique of DNA shuffling and T7 phage display. Methods Mutated homologous genes of bovine, mouse and human were digested with DNase Ⅰ , for DNA shuffling, and and DNA about 20 - 50 bp was extracted from the gel. These DNA fragments were used mutants about 500 bp were obtained. After digested with restriction enzyme, the mutants about 500 bp were ligated to T7 phage vector, the product of which was packaged with a T7 packaging extracts. Recombinant phage library was amplified and screened by competitive bio-panning method. After 5 rounds of panning, high MIF-binding phages were enriched. Plaques were made and 20 single clones were chosen to be sequenced. The result of sequencing was analyzed by bio-informatics software. The competitive ELISA was carried out to check whether the affinity of phage to MIF had been improved. Results Twenty positive single phage clones have a single consistent sequence, indicating that they are derived from a single clone. This clone is named sHCD74m, while its gene is named sHCD74mD. The result of competitive ELISA showed that sHCD74m prevents natural sCD74 from binding MIF. Conclusion sHCD74mD, the gene of sCD74 mutant that binds MIF with high affinity, has been made.

关 键 词：DNA SHUFFLING 巨噬细胞移动抑制因子 CD74 噬菌体展示技术 MHC Ⅱ恒定链 

分 类 号：Q513[生物学—生物化学] Q939.48