重组大鼠溶菌酶C端多肽的原核表达与纯化  

Prokaryotic expression and purification of lysozyme C - terminal fragment in recombinant rat

作　　者：王利娟[1] 刘誉[2] 

机构地区：[1]华南农业大学生命科学学院,510642 [2]暨南大学医学院

出　　处：《中国医学创新》2010年第19期5-8,共4页Medical Innovation of China

摘　　要：目的 应用基因重组技术,构建pET30a（＋）与LY70的原核表达重组质粒.方法 采用PCR方法扩增LY70基因,经Nde I和Not I双酶切后插入相同酶切pET30a（＋）载体,转化DH5α感受态细胞并进行阳性克隆筛选,用限制性内切酶酶切和核酸序列检测方法鉴定重组质粒DNA,将其转入宿主菌Ecoli Rosetta （DE3）,用IPTG对重组质粒进行诱导表达,SDS-PAGE及Western blot方法鉴定表达蛋白的分子质量及特异性,表达产物经Ni＋-NTA琼脂糖柱层析纯化.结果 成功构建了重组表达载体pET30a（＋）与LY70,并优化表达,表达产物主要以可溶性蛋白的形式存在.结论 原核细胞可以高效表达溶菌酶的C端多肽而不影响原核表达系统,不失为一种制备溶菌酶功能性多肽的有效手段.Objective To explore the feasibility that LY70 gene was amplified by PCR and cloned into pET - 30a（ ＋ ）. Methods The amplified fragment was inserted into the plasmid pET30a（ ＋ ） that was digested with Nde I and Not I. The recombinant plasmid pET30/LY70 was transformed into E. coli DH5a. The recombinant plasmids were successfully introduced into E. coli Rosetta （ DE3 ） and were induced by IPTG. SDS - PAGE and Western blot analysis were used to detect the confusion protein. The recombinant plasmids were identified and confirmed with enzymedigestion and sequencing. Results The expression products were purified by using Ni ＋ - NTA agarose chromatography. Conclusion Prokaryotic cells can express high C terminal peptide of lysozyme without influence on prokaryotic expression system.

关 键 词：溶菌酶 原核表达 重组多肽 纯化 

分 类 号：R394[医药卫生—医学遗传学]