噬菌体纳米抗体展示库构建中电转化条件的优化  

Optimization of Electrotransformation Conditions in Construction of Phage Nanobody Display Library

作　　者：李艳宁 李光琪 屈昱良[1] 潘俊斐 楚元奎[1] 张晓春[2] 徐广贤[1,3] LI Yanning;LI Guangqi;QU Yuliang;PAN Junfei;CHU Yuankui;ZHANG Xiaochun;XU Guangxian(School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Department of Pediatrics,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Department of Medical Laboratory,School of Medical Technology,Guangdong Medical University,Dongguan 523000,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏医科大学总医院儿科,银川750004 [3]广东医科大学医学技术学院医学检验系,东莞523000

出　　处：《宁夏医科大学学报》2021年第5期481-485,共5页Journal of Ningxia Medical University

基　　金：宁夏重点研发计划(社发领域)重点项目(2018BEG02002)。

摘　　要：目的对DNA电转化条件进行优化,以达到高的转化效率。方法测定不同生长阶段的大肠杆菌TG1、不同电压、不同外源基因(噬菌体质粒pCANTAB5e与驼源VHH片段的连接产物)质量、T4 DNA连接酶去除前后及转化孵育后不同培养温度等条件下的电转化效率,寻找最适电转化条件。结果当大肠杆菌TG1处于生长对数期(OD600为0.4左右)时制备电感受态细胞,取用乙醇沉淀法去除T4 DNA连接酶后的0.7μg连接产物,在电压2.3 kV、电容25μF、电阻200Ω的条件下,采用0.2 cm的电击杯进行电转化,将获得高的转化效率,达7.9×10^(7)CFU/μg DNA,而转化后的培养温度(30℃和37℃)对电转化效率并没有明显影响。结论经优化电转化条件后得到了高的电转化效率,为构建抗体库奠定基础。Objective To achieve high transformation efficiency by optimizing the DNA electrotransformation conditions.Methods The optimal conditions of electroporation were explored on the different factors,such as concentration of E.coli TG1,voltage,foreign gene quality(the ligation products of phage plasmid pCANTAB5e and cameled VHH fragment),impacts of T4 DNA ligase,and different culture temperatures.Results Electrocompetent cells were prepared when E.coli TG1 was in the logarithmic growth phase(OD600 was about 0.4).After removing the T4 DNA ligase by ethanol precipitation,0.7μg of the ligation products was got,and a 0.2 cm electric shock cup was used for electric conversion in the condition of 2.3 kV,25μF,200Ωwill obtain a higher conversion efficiency,reaching 7.9×10^(7)CFU/μg DNA,and the culture temperature(30℃and 37℃)had no affect on the electrotransformation efficiency.Conclusion After optimizing the electrotransformation conditions,high electrotransformation efficiency was obtained,which laid the foundation for the construction of phage nanobody display libraries.

关 键 词：噬菌体抗体库 电转化 转化效率 

分 类 号：Q93[生物学—微生物学]