人非肌肉肌球蛋白轻链激酶N端表达载体的构建及其在大肠杆菌中的表达

Construction of pBK- human non-muscle MLCK N-terminal and its expression in E. coli XL1-blue

目的 构建人非肌肉肌球蛋白轻链激酶 (hnmML CK)N端表达载体及其体外表达与表达产物的纯化。方法 用PCR方法扩增hnmMLCK的N端cDNA ,插入质粒 pBK cmv中构建成表达载体 ,转化入大肠杆菌XL1 blue ,经IPTG诱导表达 ,表达产物用电洗脱的方法初步纯化。结果 经限制性内切酶酶切图谱分析和DNA序列测定证明所构建质粒为hnmMLCK重组质粒 ,SDS PAGE证实获得分子量为 2 1ku的融合蛋白 ,表达量约占菌体总蛋白的 2 1%。结论 成功构建了重组hnmMLCK表达载体 ,并在大肠杆菌中获得表达 。

Objective To construct the recombinant vector for expression of human non-muscle myosin light chain kinase (hnmMLCK) N-terminal in E.coli. Methods PCR was used to amplify hnmMLCK N-terminal cDNA, which was inserted into pBK-cmv to construct expression vector. The recombinant vector was transformed into XL1-blue and IPTG was used to induce expression. SDS-PAGE and electroelude were used to purify expressed product. Results Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the constructed plasmid was the recombinant one. The expressed protein was purified by electroelude, and the quantity is about 21% of the whole protein. Conclusion The expressed vector is successfully constructed and hnmMLCK expressed in E.coli, which lay a good basis for manufacture and clinical application of the enzyme.